Extracellular matrix compositions for the treatment of cancer

ABSTRACT

The present invention is directed to methods of inhibiting cancer cell growth or proliferation by contacting the cancer cell with an extracellular matrix (ECM) composition. Also provided are methods of delivering a chemotherapeutic agent to a cancer cell by contacting a cancer cell with an extracellular matrix composition containing a chemotherapeutic agent. Also provided are compositions containing ECM and a chemotherapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/362,261 filed Nov. 28, 2016, now pending; which is a continuationapplication of U.S. application Ser. No. 14/711,646 filed May 13, 2015,now issued as U.S. Pat. No. 9,506,038; which is a continuationapplication of U.S. application Ser. No. 12/822,126 filed Jun. 23, 2010,now issued as U.S. Pat. No. 9,034,312; which is a continuation-in-partapplication of U.S. application Ser. No. 12/363,479 filed Jan. 30, 2009,now issued as U.S. Pat. No. 8,852,637, which claims the benefit under 35U.S.C. § 119(e) to U.S. Application Ser. No. 61/114,966 filed Nov. 14,2008, now expired. The disclosure of each of the prior applications isconsidered part of and is incorporated by reference in the disclosure ofthis application.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to use of extracellular matrixcompositions and more specifically, the use of extracellular matrixcompositions to inhibit cell proliferation.

Background Information

The extracellular matrix (ECM) is a complex structural entitysurrounding and supporting cells that are found in vivo within mammaliantissues. The ECM is often referred to as the connective tissue. The ECMis primarily composed of three major classes of biomolecules includingstructural proteins such as collagens and elastins, specialized proteinssuch as fibrillins, fibronectins, and laminins, and proteoglycans.

Growth of ECM compositions in vitro and their use in a variety oftherapeutic and medical applications have been described in the art. Onetherapeutic application of such ECM compositions includes treatment andrepair of soft tissue and skin defects such as wrinkles and scars.

The repair or augmentation of soft tissue defects caused by defects,such as, acne, surgical scarring or aging has proven to be verydifficult. A number of materials have been used to correct soft tissuedefects with varying degrees of success, however, no material has beencompletely safe and effective. For example, silicon causes a variety ofphysiological and clinical problems including long term side effects,such as nodules, recurring cellulitis and skin ulcers.

Collagen compositions have also been used as an injectable material forsoft tissue augmentation. Collagen is the main protein of connectivetissue and the most abundant protein in mammals, making up about 25% ofthe total protein content. There are currently 28 types of collagendescribed in literature (see, e.g., Tables 1 and 2 infra, for a detailedlisting). However, over 90% of the collagen in the body are Collagens I,II, III, and IV.

Different collagen materials have been used for treatment of soft tissuedefects, such as reconstituted injectable bovine collagen, crosslinkedcollagen, or other xenogeneic collagens. However, several problems existwith such collagens. A common problem includes the complexity and highcost of producing the implant materials to remove potentiallyimmunogenic substances to avoid allergic reactions in the subject.Additionally, treatments using such collagens have not proven longlasting.

Other materials have also been described that may be used for softtissue repair or augmentation, such as, biocompatible ceramic particlesin aqueous gels (U.S. Pat. No. 5,204,382), thermoplastic and/orthermosetting materials (U.S. Pat. No. 5,278,202), and lactic acid basedpolymer blends (U.S. Pat. No. 4,235,312). Additionally, use of naturallysecreted extracellular matrix compositions have also been described(U.S. Pat. No. 6,284,284). However, such materials have all proven tohave limitations.

Accordingly, new materials are needed for soft tissue repair andaugmentation that overcome the deficiencies of prior materials. The needexists to provide a safe, injectable, long lasting, bioabsorbable, softtissue repair and augmentation material.

In vitro cultured ECM compositions can additionally be used to treatdamaged tissue, such as, damaged cardiac muscle and related tissue. Thecompositions are useful as implants or biological coatings onimplantable devices, such as, stents or vascular prosthesis to promotevascularization in organs, such as the heart and related tissue.

Coronary heart disease (CHD), also called coronary artery disease (CAD),ischaemic heart disease, and atherosclerotic heart disease, ischaracterized by a narrowing of the small blood vessels that supplyblood and oxygen to the heart. Coronary heart disease is usually causedby a condition called atherosclerosis, which occurs when fatty materialand plaque builds up on the walls of arteries causing the arteries tonarrow. As the coronary arteries narrow, blood flow to the heart canslow down or stop, causing chest pain (stable angina), shortness ofbreath, heart attack, and other symptoms.

Coronary heart disease (CHD) is the leading cause of death in the UnitedStates for men and women. According to the American Heart Association,more than 15 million people have some form of the condition. While thesymptoms and signs of coronary heart disease are evident in the advancedstate of the disease, most individuals with coronary heart disease showno evidence of disease for decades as the disease progresses before asudden heart attack occurs. The disease is the most common cause ofsudden death, and is also the most common reason for death of men andwomen over 20 years of age. According to present trends in the UnitedStates, half of healthy 40-year-old males will develop CHD in thefuture, as well as one in three healthy 40-year-old women.

Current methods for improving blood flow in a diseased or otherwisedamaged heart involve invasive surgical techniques, such as, coronaryby-pass surgery, angioplasty, and endarterectomy. Such proceduresnaturally involve high-degrees of inherent risk during and aftersurgery, and often only provide a temporary remedy to cardiac ischemia.Accordingly, new treatment options are required to increase the successof currently available techniques for treating CHD and related symptoms.

In vitro cultured ECM compositions can additionally be used to repairand/or regenerate damaged cells or tissue, such as chondral orosteochondral cells. Osteochondral tissue is any tissue that relates toor contains bone or cartilage. The compositions of the present inventionare useful for treatment of osteochondral defects, such as degenerativeconnective tissue diseases, such as rheumatoid and/or osteoarthritis aswell as defects in patients who have cartilage defects due to trauma.

Current attempts at repairing osteochondral defects include implantationof human chondrocytes in biocompatible and biodegradable hydrogel graftsin attempts to improve the possibilities to restore articular cartilagelesions. Additionally, the technique of chondrocyte culture in alginatebeads or a matrix including polysulphated alginate has been described togenerate a hyaline-like cartilaginous tissue. However, attempts atrepairing endochondral lesions of articular cartilage by implantation ofhuman autologous chondrocytes have had limited success. Accordingly, newtreatment options are required to increase the success of currentlyavailable techniques for treating osteochondral defects.

In vitro cultured ECM compositions are also useful in tissue culturesystems for generation of engineered tissue implants. The field oftissue engineering involves the use of cell culture technology togenerate new biological tissues or repair damaged tissues. Fueled inpart, by the stem cell revolution, tissue engineering technology offersthe promise of tissue regeneration and replacement following trauma ortreatment of degenerative diseases. It can also be used in the contextof cosmetic procedures.

Tissue engineering techniques can be used to generate both autologousand heterologous tissue or cells using a variety of cell types andculture techniques. In creating an autologous implant, donor tissue maybe harvested and dissociated into individual cells, and subsequentlyattached and cultured on a substrate to be implanted at the desired siteof the functioning tissue. Many isolated cell types can be expanded invitro using cell culture techniques, however, anchorage dependent cellsrequire specific environments, often including the presence of athree-dimensional scaffold, to act as a template for growth.

Current tissue engineering technology provide generally, artificialimplants. Successful cell transplantation therapy depends on thedevelopment of suitable substrates for both in vitro and in vivo tissueculture. Thus the development of an extracellular matrix that containsonly natural materials and that is suitable for implantation would havemore of the characteristics of the endogenous tissue. Accordingly,generation of natural extracellular matrix material is an ongoingchallenge in the field of tissue engineering.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery thatextracellular matrix (ECM) compositions can inhibit the growth of cancercells in vivo. Accordingly, in one embodiment of the present invention,there are provided methods of inhibiting cancer cell growth orproliferation, by contacting the cancer cell with an extracellularmatrix composition as described herein.

In another embodiment of the present invention, there are providedmethods of delivering a chemotherapeutic agent to a cell or tissue,including contacting the cell or tissue with an extracellular matrixcomposition containing a chemotherapeutic agent.

In still another embodiment of the present invention there are providedcompositions containing ECM and a chemotherapeutic agent. In someembodiments, the composition further contains an immunomodulatory agent.

In yet another embodiment of the present invention there are providedmethods of inhibiting metastasis of a cancer cell by contacting thecancer cell with an extracellular matrix composition as describedherein. In various embodiments, the cancer cell is metastatic, forexample, the cancer cell may be from a metastatic tumor, such as ametastatic primary tumor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of tumor weight of B16 cells in the presence andabsence of ECM in the CAM assay.

FIG. 2 shows a plot of tumor weight of B16 cells in the presence andabsence of ECM in the CAM assay.

FIG. 3 shows a plot of tumor weight of B1 cells grown in the absence ofECM, mixed with ECM, or on ECM in the CAM assay.

FIG. 4 shows a plot of tumor weight of C6 cells in the presence andabsence of ECM, with or without cisplatin in the CAM assay.

FIG. 5 shows a plot of tumor weight of C6 cells in the presence andabsence of ECM, with or without cisplatin in the CAM assay.

FIGS. 6A and 6B show photographs of histologic analysis of C6 gliomatumors grown in the presence or absence of ECM.

FIG. 7 shows a plot of tumor weight of MDA 435 cells grown in thepresence and absence of ECM in the CAM assay.

FIG. 8 shows a photograph of tumor growth of C6 cells with no treatmentin nude mice.

FIG. 9 shows a photograph of tumor growth of C6 cells with hECMtreatment in nude mice.

FIG. 10 shows a photograph of tumor growth of C6 cells with ECM pluscisplatin treatment in nude mice.

FIG. 11 shows a photograph of tumor growth of C6 cells with cisplatintreatment in nude mice.

FIG. 12 shows a plot of the growth of C6 glioma tumors in nude mice inthe presence or absence of ECM, with or without cisplatin.

FIGS. 13A-D show photographs of tumor growth of MDA 435 cells in nudemice with no treatment (FIG. 13A), hECM treatment (FIG. 13B), hECM pluscisplatin treatment (FIG. 13C) and hECM with cisplatin treatment (FIG.13D).

FIG. 14 shows a plot of the growth of MDA 435 tumors in nude mice in thepresence or absence of ECM, with or without cisplatin.

FIG. 15 shows a photograph of tumor growth of B16 cells with (specimenon right) and without ECM (specimen on left) in rodents.

FIG. 16 shows a plot of tumor weight of B16 cells in the presence andabsence of liquid ECM in the CAM assay.

FIG. 17 shows a dose response curve of tumor weight of B16 cells grownin the presence of liquid ECM in the CAM assay.

FIG. 18 shows a plot of tumor weight of B16 cells grown in liquid ECM ofvarious molecular weight fractions in the CAM assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of using extracellular matrixcompositions for the inhibition of growth or proliferation of cancercells alone or as a biological vehicle for the delivery of achemotherapeutic agent.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

In one embodiment of the present invention, there are provided methodsof inhibiting cancer cell growth or proliferation, in which the methodincludes contacting the cancer cell with an extracellular matrixcomposition. In some embodiments, the cancer cell is metastatic. In someembodiments, the ECM may be a soluble fraction, and in other embodimentsthe ECM may be a non-soluble fraction. In still other embodiments, theECM may be a combination of soluble and non-soluble fractions.

In certain embodiments, the cancer cell is from a tumor, such as aprimary or secondary tumor. In certain embodiments, the tumor ismetastatic. In some aspects, the cancer is selected from the groupconsisting of melanoma, glioma, and adenocarcinoma. In other aspects thecancer is a cancer of the adrenal gland, bladder, bone, bone marrow,brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinaltract, stomach, colon, heart, kidney, liver, lung, muscle, ovary,pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen,testis, thymus, thyroid, or uterus. In one aspect the cancer is breastcancer; in another aspect the cancer is melanoma; and in another aspectthe cancer is colon cancer.

In another embodiment of the present invention, there are providedmethods of delivering a chemotherapeutic agent to a cancer cell, whereinthe method includes contacting a cancer cell with an extracellularmatrix composition containing a chemotherapeutic agent.

In one aspect, the contacting is in the surgical margin of the area fromwhich a tumor has been excised. In such embodiments, a tumor is removedfrom a subject using standard surgical techniques known to theclinician. ECM compositions of the invention may be placed in the spaceleft upon excision of the tumor (resection), so that the ECM is incontact with the surgical margin. In certain embodiments, the ECM mayinhibit the growth of cancer cells residing in the tumor margin. In someembodiments, the ECM composition may further contain a chemotherapeuticagent with or without an immunomodulatory agent. The use of theinvention ECM compositions in treatment of cancer following surgicalresection has the advantage of delivering localized andsustained-release delivery of a chemotherapeutic and/or immunomodulatoryagent to the tumor site. Additional benefits can include improvedhealing of surgical wounds and improved cosmetic outcome.

In still another embodiment of the present invention there are providedcompositions containing ECM and a chemotherapeutic agent. In someembodiments, the composition further contains an immunomodulatory agent.

In some embodiments the extracellular matrix compositions are generatedby culturing cells under hypoxic conditions on a two- orthree-dimensional surface in a suitable growth medium. The culturingmethod produces both soluble and non-soluble fractions which may be usedseparately or in combination to obtain physiologically acceptablecompositions having a variety of applications. In other embodiments, thecells may be cultured under standard or normal oxygen conditions.

The compositions of the present invention have a variety of applicationsincluding, but not limited to, inhibiting growth or proliferation ofcancer cells, delivery of therapeutic agents, promoting repair and/orregeneration of damaged cells or tissues, use in patches to promotetissue regeneration, use in tissue culture systems for culturing cells,such as stem cells, use in surface coatings used in association withimplantable devices (e.g., pacemakers, stents, stent grafts, vascularprostheses, heart valves, shunts, drug delivery ports or catheters),promoting soft tissue repair, augmentation, and/or improvement of a skinsurface, such as wrinkles, use as a biological anti-adhesion agent or asa biological vehicle for cell delivery or maintenance at a site ofdelivery.

The invention is based in part, on the discovery that cells cultured ontwo- or three-dimensional surfaces under conditions that stimulate theearly embryonic environment (hypoxia and reduced gravitational forces)prior to angiogenesis produces extracellular matrix compositions withfetal properties, including generation of embryonic proteins. Growth ofcells under hypoxic conditions demonstrate a unique ECM with fetalproperties and growth factor expression. Unlike the culturing of ECMunder traditional culture conditions, over 5000 genes are differentiallyexpressed in ECM cultured under hypoxic conditions. This results in acultured ECM that has different properties and a different biologicalcomposition. For example, an ECM produced under hypoxic conditions issimilar to fetal mesenchymal tissue in that it is relatively rich incollagens type III, IV, and V, and glycoproteins such as fibronectin,SPARC, thrombospondin, and hyaluronic acid.

Hypoxia also enhances expression of factors which regulate wound healingand organogenesis, such as VEGF, FGF-7, and TGF-β, as well as multipleWnt factors including Wnt's 2b, 4, 7a, 10a, and 11. Cultured embryonichuman ECM also stimulates an increase of metabolic activity in humanfibroblasts in vitro, as measured by increased enzymatic activity.Additionally, there is an increase in cell number in response to thecultured embryonic ECM.

In various embodiments, the present invention involves methods formaking extracellular matrix compositions that include one or moreembryonic proteins and applications thereof. In particular thecompositions are generated by culturing cells under hypoxic conditionson a two- or three-dimensional surface in a suitable growth medium. Thecompositions are derived by growing cells on a framework resulting in amulti-layer cell culture system. Cells grown on a framework support, inaccordance with the present invention, grow in multiple layers, forminga cellular matrix. Growth of the cultured cells under hypoxic conditionsresults in differential gene expression as the result of hypoxicculturing conditions versus traditional culture.

Extracellular matrix (ECM) is a composition of proteins and biopolymersthat substantially comprise tissue that is produced by cultivation ofcells. Stromal cells, such as fibroblasts, are an anchorage dependantcell type requiring growth while attached to materials and surfacessuitable for cell culture. The ECM materials produced by the culturedcells are deposited in a three-dimensional arrangement providing spacesfor the formation of tissue-like structures.

The cultivation materials providing three-dimensional architectures arereferred to as scaffolds. Spaces for deposition of ECM are in the formof openings within, for example woven mesh or interstitial spacescreated in a compacted configuration of spherical beads, calledmicrocarriers.

As used herein, “extracellular matrix composition” includes both solubleand non-soluble fractions or any portion or combination thereof. Thenon-soluble fraction includes those secreted extracellular matrixproteins and biological components that are deposited on the support orscaffold. The soluble fraction refers to culture media in which cellshave been cultured and into which the cells have secreted activeagent(s) and includes those proteins and biological components notdeposited on the scaffold. Both fractions may be collected, andoptionally further processed, and used individually or in combination ina variety of applications as described herein. ECM may also be obtainedfrom cadaver tissues (U.S. Pat. Nos. 6,284,284 and 6,372,494).

The soluble fraction may be concentrated or diluted to achieve anoptimal concentration. In certain embodiments, the concentration may beabout 1 mg/mL, about 5 mg/mL, about 10 mg/mL, about 50 mg/mL, about 100mg/mL, or about 200 mg/mL. The concentration may be in a range of about0.1 mg/mL to 1000 mg/mL, or about 1 to 200 mg/mL, or about 10 mg/mL to100 mg/mL.

The soluble fraction may be further fractionated according to molecularweight (e.g., by dialysis, filtration, or chromatography) so that themajority of the solute molecules in fraction meet a particular molecularweight cutoff. It will be recognized that the cutoff values are notabsolute but that a majority (e.g., 70%, 80%, 90%, or 95%) of the solutemolecules contained in a given fraction meet the molecular weightcutoff. Accordingly, in certain embodiments of the methods providedherein, the ECM composition may include a soluble fraction in which 70%,or 80%, or 90% of the solute molecules contained therein have amolecular weight of less than 5,000 Daltons, or less than 10,000Daltons, or less than 30,000 Daltons, or less than 100,000 Daltons. Inother embodiments, the ECM may include a soluble fraction in which thesolute molecules contained therein have a molecular weight of greaterthan 5,000 Daltons, or greater than 10,000 Daltons, or greater than30,000 Daltons, or greater than 100,000 Daltons. In other embodiments,the ECM may include a soluble fraction in which the solute moleculescontained therein have a molecular weight between 10,000 and 100,000Daltons, or 30,000 to 100,000 Daltons, or 10,000 to 30,000 Daltons.

The two- or three-dimensional support or scaffold used to culturestromal cells may be of any material and/or shape that: (a) allows cellsto attach to it (or can be modified to allow cells to attach to it); and(b) allows cells to grow in more than one layer (i.e., form a threedimensional tissue). In other embodiments, a substantiallytwo-dimensional sheet or membrane may be used to culture cells that aresufficiently three dimensional in form.

The biocompatible material is formed into a two- or three-dimensionalstructure or scaffold, where the structure has interstitial spaces forattachment and growth of cells into a three dimensional tissue. Theopenings and/or interstitial spaces of the framework in some embodimentsare of an appropriate size to allow the cells to stretch across theopenings or spaces. Maintaining actively growing cells stretched acrossthe framework appears to enhance production of the repertoire of growthfactors responsible for the activities described herein. If the openingsare too small, the cells may rapidly achieve confluence but be unable toeasily exit from the mesh. These trapped cells may exhibit contactinhibition and cease production of the appropriate factors necessary tosupport proliferation and maintain long term cultures. If the openingsare too large, the cells may be unable to stretch across the opening,which may lead to a decrease in stromal cell production of theappropriate factors necessary to support proliferation and maintain longterm cultures. Typically, the interstitial spaces are at least about 100um, at least 140 um, at least about 150 um, at least about 180 um, atleast about 200 um, or at least about 220 um. When using a mesh type ofmatrix, as exemplified herein, we have found that openings ranging fromabout 100 μm to about 220 μm will work satisfactorily. However,depending upon the three-dimensional structure and intricacy of theframework, other sizes are permissible. Any shape or structure thatallows the cells to stretch and continue to replicate and grow forlengthy time periods may function to elaborate the cellular factors inaccordance with the methods herein.

In some aspects, the three dimensional framework is formed from polymersor threads that are braided, woven, knitted or otherwise arranged toform a framework, such as a mesh or fabric. The materials may also beformed by casting of the material or fabrication into a foam, matrix, orsponge-like scaffold. In other aspects, the three dimensional frameworkis in the form of matted fibers made by pressing polymers or otherfibers together to generate a material with interstitial spaces. Thethree dimensional framework may take any form or geometry for the growthof cells in culture. Thus, other forms of the framework, as furtherdescribed below, may suffice for generating the appropriate conditionedmedium.

A number of different materials may be used to form the scaffold orframework. These materials include non-polymeric and polymericmaterials. Polymers, when used, may be any type of polymer, such ashomopolymers, random polymers, copolymers, block polymers, coblockpolymers (e.g., di, tri, etc.), linear or branched polymers, andcrosslinked or non-crosslinked polymers. Non-limiting examples ofmaterials for use as scaffolds or frameworks include, among others,glass fibers, polyethylenes, polypropylenes, polyamides (e.g., nylon),polyesters (e.g., dacron), polystyrenes, polyacrylates, polyvinylcompounds (e.g., polyvinylchloride; PVC), polycarbonates,polytetrafluorethylenes (PTFE; TEFLON), thermanox (TPX), nitrocellulose,polysaccharides (e.g., celluloses, chitosan, agarose), polypeptides(e.g., silk, gelatin, collagen), polyglycolic acid (PGA), and dextran.

In some aspects, the framework may be made of materials that degradeover time under the conditions of use. Biodegradable also refers toabsorbability or degradation of a compound or composition whenadministered in vivo or under in vitro conditions. Biodegradation mayoccur through the action of biological agents, either directly orindirectly. Non-limiting examples of biodegradable materials include,among others, polylactide, polyglycolide, poly(trimethylene carbonate),poly(lactide-co-glycolide) (i.e., PLGA), polyethylene terephtalate(PET), polycaprolactone, catgut suture material, collagen (e.g., equinecollagen foam), polylactic acid, or hyaluronic acid. For example, thesematerials may be woven into a three-dimensional framework such as acollagen sponge or collagen gel.

In other aspects, where the cultures are to be maintained for longperiods of time, cryopreserved, and/or where additional structuralintegrity is desired, the three dimensional framework may be comprisedof a nonbiodegradable material. As used herein, a nonbiodegradablematerial refers to a material that does not degrade or decomposesignificantly under the conditions in the culture medium. Exemplarynondegradable materials include, as non-limiting examples, nylon,dacron, polystyrene, polyacrylates, polyvinyls, polytetrafluoroethylenes(PTFE), expanded PTFE (ePTFE), and cellulose. An exemplary nondegradingthree dimensional framework comprises a nylon mesh, available under thetradename Nitex®, a nylon filtration mesh having an average pore size of140 μm and an average nylon fiber diameter of 90 μm (#3-210/36, Tetko,Inc., N.Y.).

In other aspects, the scaffold or framework is a combination ofbiodegradable and non-biodegradable materials. The non-biodegradablematerial provides stability to the scaffold during culturing while thebiodegradable material allows formation of interstitial spacessufficient for generating cell networks that produce the cellularfactors sufficient for therapeutic applications. The biodegradablematerial may be coated onto the non-biodegradable material or woven,braided or formed into a mesh. Various combinations of biodegradable andnon-biodegradable materials may be used. An exemplary combination ispoly(ethylene therephtalate) (PET) fabrics coated with a thinbiodegradable polymer film, poly[D-L-lactic-co-glycolic acid), in orderto obtain a polar structure.

In various aspects, the scaffold or framework material may bepre-treated prior to inoculation with cells to enhance cell attachment.For example, prior to inoculation with cells, nylon screens in someembodiments are treated with 0.1 M acetic acid, and incubated inpolylysine, fetal bovine serum, and/or collagen to coat the nylon.Polystyrene could be similarly treated using sulfuric acid. In otherembodiments, the growth of cells in the presence of thethree-dimensional support framework may be further enhanced by adding tothe framework or coating it with proteins (e.g., collagens, elastinfibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g.,heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatansulfate, keratan sulfate, etc.), fibronectins, and/or glycopolymer(poly[N-p-vinylbenzyl-D-lactoamide], PVLA) in order to improve cellattachment. Treatment of the scaffold or framework is useful where thematerial is a poor substrate for the attachment of cells.

In one aspect, mesh is used for production of ECM. The mesh is a wovennylon 6 material in a plain weave form with approximately 100 μmopenings and approximately 125 μm thick. In culture, fibroblast cellsattach to the nylon through charged protein interactions and grow intothe voids of the mesh while producing and depositing ECM proteins. Meshopenings that are excessively large or small may not be effective butcould differ from those above without substantially altering the abilityto produce or deposit ECM. In another aspect, other woven materials areused for ECM production, such as polyolefin's, in weave configurationsgiving adequate geometry for cell growth and ECM deposition.

For example, nylon mesh is prepared for cultivation in any of the stepsof the invention by cutting to the desired size, washing with 0.1-0.5Macetic acid followed by rinsing with high purity water and then steamsterilized. For use as a three-dimensional scaffold for ECM productionthe mesh is sized into squares approximately 10 cm×10 cm. However, themesh could be any size appropriate to the intended application and maybe used in any of the methods of the present invention, includingcultivation methods for inoculation, cell growth and ECM production andpreparation of the final form.

In other aspects, the scaffold or framework for generating the culturedthree dimensional tissues is composed of microcarriers, which are beadsor particles. The beads may be microscopic or macroscopic and mayfurther be dimensioned so as to permit penetration into tissues orcompacted to form a particular geometry. In some tissue penetratingembodiments, the framework for the cell cultures comprises particlesthat, in combination with the cells, form a three dimensional tissue.The cells attach to the particles and to each other to form a threedimensional tissue. The complex of the particles and cells is ofsufficient size to be administered into tissues or organs, such as byinjection or catheter.

As used herein, a “microcarrier” refers to a particle having size ofnanometers to micrometers, where the particles may be any shape orgeometry, being irregular, non-spherical, spherical, or ellipsoid.Typically growth on beads or microcarriers is referred to as atwo-dimensional system or scaffold or framework.

The size of the microcarriers suitable for the purposes herein can be ofany size suitable for the particular application. In some embodiments,the size of microcarriers suitable for the three dimensional tissues maybe those administrable by injection. In some embodiments, themicrocarriers have a particle size range of at least about 1 μm, atleast about 10 μm, at least about 25 μm, at least about 50 μm, at leastabout 100 μm, at least about 200 μm, at least about 300 μm, at leastabout 400 μm, at least about 500 μm, at least about 600 μm, at leastabout 700 μm, at least about 800 μm, at least about 900 μm, at leastabout 1000 μm.

In some aspects in which the microcarriers are made of biodegradablematerials. In some aspects, microcarriers comprising two or more layersof different biodegradable polymers may be used. In some embodiments, atleast an outer first layer has biodegradable properties for forming thethree dimensional tissues in culture, while at least a biodegradableinner second layer, with properties different from the first layer, ismade to erode when administered into a tissue or organ.

In some aspects, the microcarriers are porous microcarriers. Porousmicrocarriers refer to microcarriers having interstices through whichmolecules may diffuse in or out from the microparticle. In otherembodiments, the microcarriers are non-porous microcarriers. A nonporousmicroparticle refers to a microparticle in which molecules of a selectsize do not diffuse in or out of the microparticle.

Microcarriers for use in the compositions are biocompatible and have lowor no toxicity to cells. Suitable microcarriers may be chosen dependingon the tissue to be treated, type of damage to be treated, the length oftreatment desired, longevity of the cell culture in vivo, and timerequired to form the three dimensional tissues. The microcarriers maycomprise various polymers, natural or synthetic, charged (i.e., anionicor cationic) or uncharged, biodegradable, or non-biodegradable. Thepolymers may be homopolymers, random copolymers, block copolymers, graftcopolymers, and branched polymers.

In some aspects, the microcarriers comprise non-biodegradablemicrocarriers. Non-biodegradable microcapsules and microcarriersinclude, but not limited to, those made of polysulfones,poly(acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate,hydroxyethylmethacrylate-methyl-methacrylate copolymers. These areuseful to provide tissue bulking properties or in embodiments where themicrocarriers are eliminated by the body.

In some aspects, the microcarriers comprise degradable scaffolds. Theseinclude microcarriers made from naturally occurring polymers,non-limiting example of which include, among others, fibrin, casein,serum albumin, collagen, gelatin, lecithin, chitosan, alginate orpoly-amino acids such as poly-lysine. In other aspects, the degradablemicrocarriers are made of synthetic polymers, non-limiting examples ofwhich include, among others, polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polydioxanonetrimethylene carbonate, polyhybroxyalkonates (e.g.,poly(hydroxybutyrate), poly(ethyl glutamate), poly(DTHiminocarbony(bisphenol A iminocarbonate), poly(ortho ester), andpolycyanoacrylates.

In some aspects, the microcarriers comprise hydrogels, which aretypically hydrophilic polymer networks filled with water. Hydrogels havethe advantage of selective trigger of polymer swelling. Depending on thecomposition of the polymer network, swelling of the microparticle may betriggered by a variety of stimuli, including pH, ionic strength,thermal, electrical, ultrasound, and enzyme activities. Non-limitingexamples of polymers useful in hydrogel compositions include, amongothers, those formed from polymers of poly(lactide-co-glycolide);poly(N-isopropylacrylamide); poly(methacrylic acid-g-polyethyleneglycol); polyacrylic acid and poly(oxypropylene-co-oxyethylene) glycol;and natural compounds such as chrondroitan sulfate, chitosan, gelatin,fibrinogen, or mixtures of synthetic and natural polymers, for examplechitosan-poly (ethylene oxide). The polymers may be crosslinkedreversibly or irreversibly to form gels adaptable for forming threedimensional tissues.

In accordance with the present invention the culturing method isapplicable to proliferation of different types of cells, includingstromal cells, such as fibroblasts, and particularly primary humanneonatal foreskin fibroblasts. In various aspects, the cells inoculatedonto the framework can be stromal cells comprising fibroblasts, with orwithout other cells, as further described below. In some embodiments,the cells are stromal cells that are typically derived from connectivetissue, including, but not limited to: (1) bone; (2) loose connectivetissue, including collagen and elastin; (3) the fibrous connectivetissue that forms ligaments and tendons, (4) cartilage; (5) theextracellular matrix of blood; (6) adipose tissue, which comprisesadipocytes; and (7) fibroblasts.

Stromal cells can be derived from various tissues or organs, such asskin, heart, blood vessels, bone marrow, skeletal muscle, liver,pancreas, brain, foreskin, which can be obtained by biopsy (whereappropriate) or upon autopsy. In one aspect, fetal fibroblasts can beobtained in high quantity from foreskin, such as neonatal foreskins.

In some aspects, the cells comprise fibroblasts, which can be from afetal, neonatal, adult origin, or a combination thereof. In someaspects, the stromal cells comprise fetal fibroblasts, which can supportthe growth of a variety of different cells and/or tissues. As usedherein, a fetal fibroblast refers to fibroblasts derived from fetalsources. As used herein, neonatal fibroblast refers to fibroblastsderived from newborn sources. Under appropriate conditions, fibroblastscan give rise to other cells, such as bone cells, fat cells, and smoothmuscle cells and other cells of mesodermal origin. In some embodiments,the fibroblasts comprise dermal fibroblasts, which are fibroblastsderived from skin. Normal human dermal fibroblasts can be isolated fromneonatal foreskin. These cells are typically cryopreserved at the end ofthe primary culture.

In other aspects, the three-dimensional tissue can be made using stem orprogenitor cells, either alone, or in combination with any of the celltypes discussed herein. Stem and progenitor cells include, by way ofexample and not limitation, embryonic stem cells, hematopoietic stemcells, neuronal stem cells, epidermal stem cells, and mesenchymal stemcells.

In some embodiments, a “specific” three-dimensional tissue can beprepared by inoculating the scaffold with cells derived from aparticular organ, i.e., skin, heart, and/or from a particular individualwho is later to receive the cells and/or tissues grown in culture inaccordance with the methods described herein.

For certain uses in vivo it is preferable to obtain the stromal cellsfrom the patient's own tissues. The growth of cells in the presence ofthe stromal support framework can be further enhanced by adding to theframework, or coating the framework support with proteins, e.g.,collagens, laminins, elastic fibers, reticular fibers, glycoproteins;glycosaminoglycans, e.g., heparan sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.; acellular matrix, and/or other materials.

Thus, since the two- or three-dimensional culture system describedherein is suitable for growth of diverse cell types and tissues, anddepending upon the tissue to be cultured and the collagen types desired,the appropriate stromal cells may be selected to inoculate theframework.

While the methods and applications of the present invention are suitablefor use with different cell types, such as tissue specific cells ordifferent types of stromal cells as discussed herein, derivation of thecells for use with the present invention may also be species specific.Accordingly, ECM compositions may be generated that are speciesspecific. For example, the cells for use in the present invention mayinclude human cells. For example, the cells may be human fibroblasts.Likewise, the cells are from another species of animal, such as equine(horse), canine (dog) or feline (cat) cells. Additionally, cells fromone species or strain of species may be used to generate ECMcompositions for use in other species or related strains (e.g.,allogeneic, syngeneic and xenogeneic). It is also to be appreciated thatcells derived from various species may be combined to generatemulti-species ECM compositions.

Accordingly, the methods and compositions of the present invention aresuitable in applications involving non-human animals. As used herein,“veterinary” refers to the medical science concerned or connected withthe medical or surgical treatment of animals, especially domesticanimals. Common veterinary animals may include mammals, amphibians,avians, reptiles and fishes. For example, typical mammals may includedogs, cats, horses, rabbits, primates, rodents, and farm animals, suchas cows, horses, goats, sheep, and pigs.

As discussed above, additional cells may be present in the culture withthe stromal cells. These additional cells may have a number ofbeneficial effects, including, among others, supporting long term growthin culture, enhancing synthesis of growth factors, and promotingattachment of cells to the scaffold. Additional cell types include asnon-limiting examples, smooth muscle cells, cardiac muscle cells,endothelial cells, skeletal muscle cells, endothelial cells, pericytes,macrophages, monocytes, and adipocytes. Such cells may be inoculatedonto the framework along with fibroblasts, or in some aspects, in theabsence of fibroblasts. These stromal cells may be derived fromappropriate tissues or organs, including, by way of example and notlimitation, skin, heart, blood vessels, bone marrow, skeletal muscle,liver, pancreas, and brain. In other aspects, one or more other celltypes, excluding fibroblasts, are inoculated onto the scaffold. In stillother aspects, the scaffolds are inoculated only with fibroblast cells.

Fibroblasts may be readily isolated by disaggregating an appropriateorgan or tissue which is to serve as the source of the fibroblasts. Forexample, the tissue or organ can be disaggregated mechanically and/ortreated with digestive enzymes and/or chelating agents that weaken theconnections between neighboring cells making it possible to disperse thetissue into a suspension of individual cells without appreciable cellbreakage. Enzymatic dissociation can be accomplished by mincing thetissue and treating the minced tissue with any of a number of digestiveenzymes either alone or in combination. These include but are notlimited to trypsin, chymotrypsin, collagenase, elastase, hyaluronidase,DNase, pronase, and/or dispase, etc. Mechanical disruption can also beaccomplished by a number of methods including, but not limited to theuse of grinders, blenders, sieves, homogenizers, pressure cells, orinsonators to name but a few. In one aspect, excised foreskin tissue istreated using digestive enzymes, typically collagenase and/or trypsinaseto disassociate the cells from encapsulating structures.

The isolation of fibroblasts, for example, can be carried out asfollows: fresh tissue samples are thoroughly washed and minced in Hanks'balanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts can then be grown to confluency, liftedfrom the confluent culture and inoculated onto the three-dimensionalframework, see Naughton et al., 1987, J. Med. 18(3&4):219-250.Inoculation of the three-dimensional framework with a high concentrationof stromal cells, e.g., approximately 10⁶ to 5×10⁷ cells/ml, will resultin the establishment of the three-dimensional stromal support in shorterperiods of time.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thefibroblasts and/or other stromal cells and/or elements can be obtained.This also may be accomplished using standard techniques for cellseparation including, but not limited to, cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counter-streaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. For a review of clonal selection and cell separationtechniques, see Freshney, Culture of Animal Cells. A Manual of BasicTechniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-168.

In one aspect, isolated fibroblast cells can be grown to produce cellbanks. Cell banks are created to allow for initiating various quantitiesand timing of cultivation batches and to allow preemptive testing ofcells for contaminants and specific cellular characteristics.Fibroblasts from the cell banks are subsequently grown to increase cellnumber to appropriate levels for seeding scaffolds. Operations involvingenvironmental exposure of cells and cell contacting materials areperformed by aseptic practices to reduce the potential for contaminationof foreign materials or undesirable microbes.

In another aspect of the invention, after isolation, cells can be grownthrough several passages to a quantity suitable for building master cellbanks. The cell banks can then be, harvested and filled into appropriatevessels and preserved in cryogenic conditions. Cells in frozen vialsfrom master cell banks can be thawed and grown through additionalpassages (usually two or more). The cells can then be used to preparecryogenically preserved working cell banks.

A cell expansion step uses vials of cells at the working cell bank stageto further increase cell numbers for inoculating three-dimensionalscaffolds or supports, such as mesh or microcarriers. Each passage is aseries of sub-culture steps that include inoculating cell growthsurfaces, incubation, feeding the cells and harvesting.

Cultivation for cell banks and cell expansion can be conducted byinoculating culture vessels, such as culture flasks, roller bottles ormicrocarriers. Stromal cells, such as fibroblasts, attach to theintended growth surfaces and grow in the presence of culture media.Culture vessels, such as culture flasks, roller bottles andmicrocarriers are specifically configured for cell culture and arecommonly made from various plastic materials qualified for intendedapplications. Microcarriers typically are microscopic or macroscopicbeads and are typically made of various plastic materials. However, theycan be made from other materials such as glasses or solid/semi-solidbiologically based materials such as collagens or other materials suchas Dextran, a modified sugar complex as discussed above.

During cultivation, expended media is periodically replaced with freshmedia during the course of cell growth to maintain adequate availabilityof nutrients and removal of inhibitory products of cultivation. Cultureflasks and roller bottles provide a surface for the cells to grow ontoand are typically used for cultivation of anchorage dependent cells.

In one aspect, incubation is performed in a chamber heated at 37° C.Cultivation topologies requiring communication of media and the chamberenvironment use a 5% CO₂ v/v with air in the chamber gas space to aid inregulation of pH. Alternately, vessels equipped to maintain cultivationtemperature and pH can be used for both cell expansion and ECMproduction operations. Temperatures below 35° C. or above 38° C. and CO₂concentrations below 3% or above 12% may not be appropriate.

Harvesting cells from attachment surfaces can conducted by removal ofgrowth media and rinsing the cells with a buffered salt solution toreduce enzyme competing protein, application of disassociating enzymesthen neutralization of the enzymes after cell detachment. Harvested cellsuspension is collected and harvest fluids are separated bycentrifugation. Cell suspensions from sub-culture harvests can besampled to assess the quantity of cells recovered and other cellularattributes and are subsequently combined with fresh media and applied asinoculums. The number of passages used for preparing cell banks andscaffold inoculum is critical with regard to achieving acceptable ECMcharacteristics.

After an appropriate two- or three-dimensional scaffold is prepared, itis inoculated by seeding with the prepared stromal cells. Inoculation ofthe scaffold may be done in a variety of ways, such as sedimentation.Mesh prepared for culture of ECM under aerobic conditions are preparedin the same manner as for hypoxic grown mesh with the exception that ananaerobic chamber is not used to create hypoxic conditions.

For example, for both mesh prepared for culture of ECM under bothaerobic and hypoxic conditions, prepared and sterilized mesh is placedin sterile 150 mm diameter×15 mm deep petri dishes and stacked to athickness of approximately 10 pieces. Stacks of mesh are then inoculatedby sedimentation. Cells are added to fresh media to obtain theappropriate concentration of cells for inoculum. Inoculum is added tothe stack of mesh where cells settle onto the nylon fibers and attachwhile in incubated conditions. After an adequate time, individuallyseeded mesh sheets can be aseptically separated from the stack andplaced individually into separate 150 mm×15 mm petri dishes containingapproximately 50 ml of growth media.

Incubation of the inoculated culture is performed under hypoxicconditions, which is discovered to produce an ECM and surrounding mediawith unique properties as compared to ECM generated under normal cultureconditions. As used herein, hypoxic conditions are characterized by alower oxygen concentration as compared to the oxygen concentration ofambient air (approximately 15%-20% oxygen). In one aspect, hypoxicconditions are characterized by an oxygen concentration less than about10%. In another aspect hypoxic conditions are characterized by an oxygenconcentration of about 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to6%, 1% to 5%, 1% to 4%, 1% to 3%, or 1% to 2%. In a certain aspect, thesystem maintains about 1-3% oxygen within the culture vessel. Hypoxicconditions can be created and maintained by using a culture apparatusthat allows one to control ambient gas concentrations, for example, ananaerobic chamber.

Incubation of cell cultures is typically performed in normal atmospherewith 15-20% oxygen and 5% CO₂ for expansion and seeding, at which pointlow oxygen cultures are split to an airtight chamber that is floodedwith 95% nitrogen/5% CO₂ so that a hypoxic environment is created withinthe culture medium.

For example, petri dishes with mesh cultured for producing ECM underhypoxic conditions are initially grown in incubation at 37° C. and 95%air/5% CO₂ for 2-3 weeks. Following the period of near atmosphericcultivation, the petri dishes of mesh are incubated in a chamberdesigned for anaerobic cultivation that is purged with a gas mixture ofapproximately 95% nitrogen and 5% CO₂. Expended growth media is replacedwith fresh media at atmospheric oxygen level through the culture periodand after media is exchanged the mesh filled petri dishes are place inthe anaerobic chamber, the chamber is purged with 95% nitrogen/5% CO₂then incubated at 37° C. Cultured mesh are harvested when they reach thedesired size or contain the desire biological components.

During the incubation period, the stromal cells will grow linearly alongand envelop the three-dimensional framework before beginning to growinto the openings of the framework. The growing cells produce a myriadof growth factors, regulatory factors and proteins, some of which aresecreted in the surrounding media, and others that are deposited on thesupport to make up the extracellular matrix more fully discussed below.Growth and regulatory factors can be added to the culture, but are notnecessary. Culture of the stromal cells produces both non-soluble andsoluble fractions. The cells are grown to an appropriate degree to allowfor adequate deposition of extracellular matrix proteins.

During culturing of the three-dimensional tissues, proliferating cellsmay be released from the framework and stick to the walls of the culturevessel where they may continue to proliferate and form a confluentmonolayer. To minimize this occurrence, which may affect the growth ofcells, released cells may be removed during feeding or by transferringthe cell culture to a new culture vessel. Removal of the confluentmonolayer or transfer of the cultured tissue to fresh media in a newvessel maintains or restores proliferative activity of the cultures. Insome aspects, removal or transfers may be done in a culture vessel whichhas a monolayer of cultured cells exceeding 25% confluency.Alternatively, the culture in some embodiments is agitated to preventthe released cells from sticking; in others, fresh media is infusedcontinuously through the system. In some aspects, two or more cell typescan be cultured together either at the same time or one first followedby the second (e.g., fibroblasts and smooth muscle cells or endothelialcells).

After inoculation of the scaffolds, the cell culture is incubated in anappropriate nutrient medium and incubation conditions that supportsgrowth of cells into the three dimensional tissues. Many commerciallyavailable media such as Dulbecco's Modified Eagles Medium (DMEM), RPMI1640, Fisher's, Iscove's, and McCoy's, may be suitable for supportingthe growth of the cell cultures. The medium may be supplemented withadditional salts, carbon sources, amino acids, serum and serumcomponents, vitamins, minerals, reducing agents, buffering agents,lipids, nucleosides, antibiotics, attachment factors, and growthfactors. Formulations for different types of culture media are describedin various reference works available to the skilled artisan (e.g.,Methods for Preparation of Media, Supplements and Substrates for SerumFree Animal Cell Cultures, Alan R. Liss, New York (1984); TissueCulture: Laboratory Procedures, John Wiley & Sons, Chichester, England(1996); Culture of Animal Cells, A Manual of Basic Techniques, 4th Ed.,Wiley-Liss (2000)).

The growth or culture media used in any of the culturing steps of thepresent invention, whether under aerobic or hypoxic conditions, mayinclude serum, or be serum free. In one aspect, the media is Dulbecco'sModified Eagle Medium with 4.5 g/L glucose, alanyl-L-glutamine, Eq 2 mM,and nominally supplemented with 10% fetal bovine serum. In anotheraspect, the media is a serum free media and is Dulbecco's Modified EagleMedium with 4.5 g/L glucose base medium with Glutamax®, supplementedwith 0.5% serum albumin, 2 μg/ml heparin, 1 μg/ml recombinant basic FGF,1 μg/ml soybean trypsin inhibitor, 1× ITS supplement(insulin-transferrin-selenium, Sigma Cat. No. 13146), 1:1000 dilutedfatty acid supplement (Sigma Cat. No. 7050), and 1:1000 dilutedcholesterol. Additionally, the same media can be used for both hypoxicand aerobic cultivation. In one aspect, the growth media is changed fromserum based media to serum free media after seeding and the first weekof growth.

Incubation conditions will be under appropriate conditions of pH,temperature, and gas (e.g., O₂, CO₂, etc) to maintain an hypoxic growthcondition. In some embodiments, the cell culture can be suspended in themedium during the incubation period in order to maximize proliferativeactivity and generate factors that facilitate the desired biologicalactivities of the fractions. In addition, the culture may be “fed”periodically to remove the spent media, depopulate released cells, andadd new nutrient source. During the incubation period, the culturedcells grow linearly along and envelop the filaments of the scaffoldbefore beginning to grow into the openings of the scaffold.

During incubation under hypoxic conditions, as compared to incubationunder normal atmospheric oxygen concentrations of about 15-20%,thousands of genes are differentially expressed. Several genes have beenfound to be upregulated or downregulated in such compositions, mostnotably certain laminin species, collagen species and Wnt factors. Invarious aspects, the three dimensional extracellular matrix may bedefined by the characteristic fingerprint or suite of cellular productsproduced by the cells due to growth in hypoxic condition as comparedwith growth under normal conditions. In the extracellular matrixcompositions specifically exemplified herein, the three-dimensionaltissues and surrounding media are characterized by expression and/orsecretion of various factors.

The three dimensional tissues and compositions described herein haveextracellular matrix that is present on the scaffold or framework. Insome aspects, the extracellular matrix includes various laminin andcollagen types due to growth under hypoxic conditions and selection ofcells grown on the support. The proportions of extracellular matrix(ECM) proteins deposited can be manipulated or enhanced by selectingfibroblasts which elaborate the appropriate collagen type as well asgrowing the cells under hypoxic conditions in which expression ofspecific laminin and collagen species are upregulated or downregulated.

Selection of fibroblasts can be accomplished in some aspects usingmonoclonal antibodies of an appropriate isotype or subclass that defineparticular collagen types. In other aspects, solid substrates, such asmagnetic beads, may be used to select or eliminate cells that have boundantibody. Combination of these antibodies can be used to select(positively or negatively) the fibroblasts which express the desiredcollagen type. Alternatively, the stroma used to inoculate the frameworkcan be a mixture of cells which synthesize the desired collagen types.The distribution and origins of the exemplary type of collagen are shownin Table I.

TABLE 1 Distributions and Origins of Various Types of Collagen CollagenType Principle Tissue Distribution Cells of Origin I Loose and denseordinary Fibroblasts and reticular connective tissue; collagen cells;smooth muscle cells fibers Fibrocartilage Bone Osteoblasts DentinOdontoblasts II Hyaline and elastic cartilage Chondrocytes Vitreous bodyof eye Retinal cells III Loose connective tissue; Fibroblasts andreticular reticular fibers cells Papillary layer of dermis Smooth musclecells; endothelial cells Blood vessels IV Basement membranes Epithelialand endothelial cells Lens capsule of eye Lens fibers V Fetal membranes;placenta Fibroblasts Basement membranes Bone Smooth muscle Smooth musclecells IV Basement membranes Epithelial and endothelial cells Lenscapsule of the eye Lens fiber V Fetal membranes; placenta FibroblastsBasement membranes Bone Smooth muscle Smooth muscle cells VI Connectivetissue Fibroblasts VII Epithelial basement Fibroblasts membranesanchoring fibrils keratinocytes VIII Cornea Corneal fibroblasts IXCartilage X Hypertrophic cartilage XI Cartilage XII Papillary dermisFibroblasts XIV Reticular dermis Fibroblasts (undulin) XVII P170 bullouspemphigoid Keratinocytes antigen

Additional types of collagen that may be present in ECM compositions areshown in Table 2.

TABLE 2 Types of Collagen and Corresponding Gene(s) Collagen TypeGene(s) I COL1A1, COL1A2 II COL2A1 III COL3A1 IV COL4A1, COL4A2, COL4A3,COL4A4, COL4A5, COL4A6 V COL5A1, COL5A2, COL5A3 VI COL6A1, COL6A2,COL6A3 VII COL7A1 VIII COL8A1, COL8A2 IX COL9A1, COL9A2, COL9A3 XCOL10A1 XI COL11A1, COL11A2 XII COL12A1 XIII COL13A1 XIV COL14A1 XVCOL15A1 XVI COL16A1 XVII COL17A1 XVIII COL18A1 XIX COL19A1 XX COL20A1XXI COL21A1 XXII COL22A1 XXIII COL23A1 XXIV COL24A1 XXV COL25A1 XXVIEMID2 XXVII COL27A1 XXVIII COL28A1

As discussed above the ECM compositions described herein include variouscollagens. As shown in Table 3 of Example 1, expression of severalspecies of collagen are found to be upregulated in hypoxic cultured ECMcompositions. Accordingly, in one aspect of the present invention, theECM composition including one or more embryonic proteins, includesupregulation of collagen species as compared with that produced inoxygen conditions of about 15-20% oxygen. In another aspect, theupregulated collagen species are type V alpha 1; IX alpha 1; IX alpha 2;VI alpha 2; VIII alpha 1; IV, alpha 5; VII alpha 1; XVIII alpha 1; andXII alpha 1.

In addition to various collagens, the ECM composition described hereininclude various laminins. Laminins are a family of glycoproteinheterotrimers composed of an alpha, beta, and gamma chain subunit joinedtogether through a coiled-coil domain. To date, 5 alpha, 4 beta, and 3gamma laminin chains have been identified that can combine to form 15different isoforms. Within this structure are identifiable domains thatpossess binding activity towards other laminin and basal laminamolecules, and membrane-bound receptors. Domains VI, IVb, and IVa formglobular structures, and domains V, IIIb, and IIIa (which containcysteine-rich EGF-like elements) form rod-like structures. Domains I andII of the three chains participate in the formation of a triple-strandedcoiled-coil structure (the long arm).

Laminin chains possess shared and unique functions and are expressedwith specific temporal (developmental) and spatial (tissue-sitespecific) patterns. The laminin alpha-chains are considered to be thefunctionally important portion of the heterotrimers, as they exhibittissue-specific distribution patterns and contain the major cellinteraction sites. Vascular endothelium is known to express two lamininisoforms, with varied expression depending on the developmental stage,vessel type, and the activation state of the endothelium.

Accordingly, in one aspect of the present invention, the ECM compositionincluding one or more embryonic proteins, includes upregulation ordownregulation of various laminin species as compared with that producedin oxygen conditions of about 15-20% oxygen.

Laminin 8, is composed of alpha-4, beta-1, and gamma-1 laminin chains.The laminin alpha-4 chain is widely distributed both in adults andduring development. In adults it can be identified in the basementmembrane surrounding cardiac, skeletal, and smooth muscle fibers, and inlung alveolar septa. It is also known to exist in the endothelialbasement membrane both in capillaries and larger vessels, and in theperineurial basement membrane of peripheral nerves, as well as inintersinusoidal spaces, large arteries, and smaller arterioles of bonemarrow Laminin 8 is a major laminin isoform in the vascular endotheliumthat is expressed and adhered to by platelets and is synthesized in3T3-L1 adipocytes, with its level of synthesis shown to increase uponadipose conversion of the cells. Laminin 8 is thought to be the lamininisoform generally expressed in mesenchymal cell lineages to inducemicrovessels in connective tissues. Laminin 8 has also been identifiedin mouse bone marrow primary cell cultures, arteriolar walls, andintersinusoidal spaces where it is the major laminin isoform in thedeveloping bone marrow. Due to its localization in adult bone marrowadjacent to hematopoietic cells, laminin isoforms containing the alpha-4chain are likely to have biologically relevant interactions withdeveloping hematopoietic cells.

Accordingly, in another aspect of the present invention the ECM includesupregulation of laminin species, such as laminin 8. In another aspect,laminins produced by the three dimensional tissues of the presentinvention, includes at least laminin 8, which defines a characteristicor signature of the laminin proteins present in the composition.

The ECM compositions described herein include various Wnt factors. Wntfamily factors are signaling molecules having roles in a myriad ofcellular pathways and cell-cell interaction processes. Wnt signaling hasbeen implicated in tumorigenesis, early mesodermal patterning of theembryo, morphogenesis of the brain and kidneys, regulation of mammarygland proliferation, and Alzheimer's disease. As shown in Table 4 ofExample 1, expression of several species of Wnt proteins are found to beupregulated in hypoxic cultured ECM compositions. Accordingly, in oneaspect of the present invention, the ECM composition including one ormore embryonic proteins, includes upregulation of Wnt species ascompared with that produced in oxygen conditions of about 15-20% oxygen.In another aspect, the upregulated Wnt species are Wnt 7a and Wnt 11. Inanother aspect, Wnt factors produced by the three dimensional tissues ofthe present invention, include at least Wnt7a, and Wnt11, which definesa characteristic or signature of the Wnt proteins present in thecomposition.

A discussed throughout, the ECM compositions of the present inventionincludes both soluble and non-soluble fractions or any portion thereof.It is to be understood that the compositions of the present inventionmay include either or both fractions, as well as any combinationthereof. Additionally, individual components may be isolated from thefractions to be used individually or in combination with other isolatesor known compositions.

Accordingly, in various aspects, extracellular matrix compositionsproduced using the methods of the present invention may be used directlyor processed in various ways, the methods of which may be applicable toboth the non-soluble and soluble fractions. The soluble fraction,including the cell-free supernatant and media, may be subject tolyophilization for preserving and/or concentrating the factors. Variousbiocompatible preservatives, cryoprotectives, and stabilizer agents maybe used to preserve activity where required. Examples of biocompatibleagents include, among others, glycerol, dimethyl sulfoxide, andtrehalose. The lyophilizate may also have one or more excipients such asbuffers, bulking agents, and tonicity modifiers. The freeze-dried mediamay be reconstituted by addition of a suitable solution orpharmaceutical diluent, as further described below.

In other aspects, the soluble fraction is dialyzed. Dialysis is one ofthe most commonly used techniques to separate sample components based onselective diffusion across a porous membrane. The pore size determinesmolecular-weight cutoff (MWCO) of the membrane that is characterized bythe molecular-weight at which 90% of the solute is retained by themembrane. In certain aspects membranes with any pore size iscontemplated depending on the desired cutoff. Typical cutoffs are 5,000Daltons, 10,000 Daltons, 30,000 Daltons, and 100,000 Daltons, howeverall sizes are contemplated.

In some aspects, the soluble fraction may be processed by precipitatingthe active components (e.g., growth factors) in the media. Precipitationmay use various procedures, such as salting out with ammonium sulfate oruse of hydrophilic polymers, for example polyethylene glycol.

In other aspects, the soluble fraction is subject to filtration usingvarious selective filters. Processing the soluble fraction by filteringis useful in concentrating the factors present in the fraction and alsoremoving small molecules and solutes used in the soluble fraction.Filters with selectivity for specified molecular weights include <5000Daltons, <10,000 Daltons, and <15,000 Daltons. Other filters may be usedand the processed media assayed for therapeutic activity as describedherein. Exemplary filters and concentrator system include those basedon, among others, hollow fiber filters, filter disks, and filter probes(see, e.g., Amicon Stirred Ultrafiltration Cells).

In still other aspects, the soluble fraction is subject tochromatography to remove salts, impurities, or fractionate variouscomponents of the medium. Various chromatographic techniques may beemployed, such as molecular sieving, ion exchange, reverse phase, andaffinity chromatographic techniques. For processing conditioned mediumwithout significant loss of bioactivity, mild chromatographic media maybe used. Non-limiting examples include, among others, dextran, agarose,polyacrylamide based separation media (e.g., available under varioustradenames, such as Sephadex, Sepharose, and Sephacryl).

In still other aspects, the conditioned media is formulated asliposomes. The growth factors may be introduced or encapsulated into thelumen of liposomes for delivery and for extending life time of theactive factors. As known in the art, liposomes can be categorized intovarious types: multilamellar (MLV), stable plurilamellar (SPLV), smallunilamellar (SUV) or large unilamellar (LUV) vesicles. Liposomes can beprepared from various lipid compounds, which may be synthetic ornaturally occurring, including phosphatidyl ethers and esters, such asphosphotidylserine, phosphotidylcholine, phosphatidyl ethanolamine,phosphatidylinositol, dimyristoylphosphatidylcholine; steroids such ascholesterol; cerebrosides; sphingomyelin; glycerolipids; and otherlipids (see, e.g., U.S. Pat. No. 5,833,948).

The soluble fraction may be used directly without additional additives,or prepared as pharmaceutical compositions with various pharmaceuticallyacceptable excipients, vehicles or carriers. A “pharmaceuticalcomposition” refers to a form of the soluble and/or non-solublefractions and at least one pharmaceutically acceptable vehicle, carrier,or excipient. For intradermal, subcutaneous or intramuscularadministration, the compositions may be prepared in sterile suspension,solutions or emulsions of the extracellular matrix compositions inaqueous or oily vehicles. The compositions may also contain formulatingagents, such as suspending, stabilizing or dispersing agents.Formulations for injection may be presented in unit dosage form, ampulesin multidose containers, with or without preservatives. Alternatively,the compositions may be presented in powder form for reconstitution witha suitable vehicle including, by way of example and not limitation,sterile pyrogen free water, saline, buffer, or dextrose solution.

In other aspects, the three dimensional tissues are cryopreservedpreparations, which are thawed prior to use. Pharmaceutically acceptablecryopreservatives include, among others, glycerol, saccharides, polyols,methylcellulose, and dimethyl sulfoxide. Saccharide agents includemonosaccharides, disaccharides, and other oligosaccharides with glasstransition temperature of the maximally freeze-concentrated solution(Tg) that is at least −60,−50,−40,−30,−20,−10, or 0° C. An exemplarysaccharide for use in cryopreservation is trehalose.

In some aspects, the three dimensional tissues are treated to kill thecells prior to use. In some aspects, the extracellular matrix depositedon the scaffolds may be collected and processed for administration (seeU.S. Pat. Nos. 5,830,708 and 6,280,284, incorporated herein byreference).

In other embodiments, the three dimensional tissue may be concentratedand washed with a pharmaceutically acceptable medium for administration.Various techniques for concentrating the compositions are available inthe art, such as centrifugation or filtering. Examples include, dextransedimentation and differential centrifugation. Formulation of the threedimensional tissues may also involve adjusting the ionic strength of thesuspension to isotonicity (i.e., about 0.1 to 0.2) and to physiologicalpH (i.e., pH 6.8 to 7.5). The formulation may also contain lubricants orother excipients to aid in administration or stability of the cellsuspension. These include, among others, saccharides (e.g., maltose) andorganic polymers, such as polyethylene glycol and hyaluronic acid.Additional details for preparation of various formulations are describedin U.S. Patent Publication No. 2002/0038152, incorporated herein byreference.

As discussed above, the ECM compositions of the present invention may beprocessed in a number of ways depending on the anticipated applicationand appropriate delivery or administration of the ECM composition. Forexample, the compositions may be delivered as three-dimensionalscaffolds or implants, or the compositions may be formulated forinjection as described above. The terms “administration” or“administering” are defined to include an act of providing a compound orpharmaceutical composition of the invention to a subject in need oftreatment. The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. The phrases “systemic administration,”“administered systemically,” “peripheral administration” and“administered peripherally” as used herein mean the administration of acompound, drug or other material other than directly into the centralnervous system, such that it enters the subject's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration.

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally the subject is human,although as will be appreciated by those in the art, the subject may bean animal. Thus other animals, including mammals such as rodents(including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits,farm animals including cows, horses, goats, sheep, pigs, etc., andprimates (including monkeys, chimpanzees, orangutans and gorillas) areincluded within the definition of subject.

The extracellular matrix compositions of the present invention have avariety of applications including, but not limited to, promoting repairand/or regeneration of damaged cells or tissues, use in patches topromote tissue regeneration, use in tissue culture systems for culturingcells, such as stem cells, use in surface coatings used in associationwith implantable devices (e.g., pacemakers, stents, stent grafts,vascular prostheses, heart valves, shunts, drug delivery ports orcatheters), promoting soft tissue repair, augmentation, and/orimprovement of a skin surface, such as wrinkles, use as a biologicalanti-adhesion agent or as a biological vehicle for cell delivery ormaintenance at a site of delivery.

Additionally, the ECM compositions derived from culturing cells asdescribed in any method herein, may be used in any other application ormethod of the present invention. For example, the ECM compositionsgenerated by culturing cells using the tissue culture system of thepresent invention may be used, for example, in the repair and/orregeneration of cells, use in patches to promote tissue regeneration,use in tissue culture systems for culturing cells, such as stem cells,use in surface coatings used in association with implantable devices(e.g., pacemakers, stents, stent grafts, vascular prostheses, heartvalves, shunts, drug delivery ports or catheters), promoting soft tissuerepair, augmentation, and/or improvement of a skin surface, such aswrinkles, use as a biological anti-adhesion agent or as a biologicalvehicle for cell delivery or maintenance at a site of delivery.

In various embodiments, the present invention includes methods forrepair and/or regeneration of cells or tissue and promoting soft tissuerepair. One embodiment includes a method of repair and/or regenerationof cells by contacting cells to be repaired or regenerated with theextracellular matrix compositions of the present invention. The methodmay be used for repair and/or regeneration of a variety of cells asdiscussed herein, including osteochondral cells.

In one aspect, the method contemplates repair of osteochondral defects.As used herein, “osteochondral cells” refers to cells which belong toeither the chondrogenic or osteogenic lineage or which can undergodifferentiation into either the chondrogenic or osteogenic lineage,depending on the environmental signals. This potential can be tested invitro or in vivo by known techniques. Thus, in one aspect, the ECMcompositions of the present invention are used to repair and/orregenerate, chondrogenic cells, for example, cells which are capable ofproducing cartilage or cells which themselves differentiate into cellsproducing cartilage, including chondrocytes and cells which themselvesdifferentiate into chondrocytes (e.g., chondrocyte precursor cells).Thus, in another aspect, the compositions of the present invention areuseful in repair and/or regeneration of connective tissue. As usedherein, “connective tissue” refers to any of a number of structuraltissues in the body of a mammal including but not limited to bone,cartilage, ligament, tendon, meniscus, dermis, hypodermis, muscle, fattytissue, joint capsule.

The ECM compositions of the present invention may be used for treatingosteochondral defects of a diarthroidal joint, such as knee, an ankle,an elbow, a hip, a wrist, a knuckle of either a finger or toe, or atemperomandibular joint. Such osteochondral defects can be the result oftraumatic injury (e.g., a sports injury or excessive wear) or a diseasesuch as osteoarthritis. A particular aspect relates to the use of thematrix of the present invention in the treatment or prevention ofsuperficial lesions of osteoarthritic cartilage. Additionally thepresent invention is of use in the treatment or prevention ofosteochondral defects which result from ageing or from giving birth.Osteochondral defects in the context of the present invention shouldalso be understood to comprise those conditions where repair ofcartilage and/or bone is required in the context of surgery such as, butnot limited to, cosmetic surgery (e.g., nose, ear). Thus such defectscan occur anywhere in the body where cartilage or bone formation isdisrupted or where cartilage or bone are damaged or non-existent due toa genetic defect.

As discussed above, growth factors or other biological agents whichinduce or stimulate growth of particular cells may be included in theECM compositions of the present invention. The type of growth factorswill be dependent on the cell-type and application for which thecomposition is intended. For example, in the case of osteochondralcells, additional bioactive agents may be present such as cellulargrowth factors (e.g., TGF-β), substances that stimulate chondrogenesis(e.g., BMPs that stimulate cartilage formation such as BMP-2, BMP-12 andBMP-13), factors that stimulate migration of stromal cells to thescaffold, factors that stimulate matrix deposition, anti-inflammatories(e.g., non-steroidal anti-inflammatories), immunosuppressants (e.g.,cyclosporins). Other proteins may also be included, such as other growthfactors such as platelet derived growth factors (PDGF), insulin-likegrowth factors (IGF), fibroblast growth factors (FGF), epidermal growthfactor (EGF), human endothelial cell growth factor (ECGF), granulocytemacrophage colony stimulating factor (GM-CSF), vascular endothelialgrowth factor (VEGF), cartilage derived morphogenetic protein (CDMP),other bone morphogenetic proteins such as OP-1, OP-2, BMP3, BMP4, BMP9,BMP11, BMP14, DPP, Vg-1, 60A, and Vgr-1, collagens, elastic fibers,reticular fibers, glycoproteins or glycosaminoglycans, such as heparinsulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate,keratin sulfate, etc. For example, growth factors such as TGF-β, withascorbate, have been found to trigger chondrocyte differentiation andcartilage formation by chondrocytes. In addition, hyaluronic acid is agood substrate for the attachment of chondrocytes and other stromalcells and can be incorporated as part of the scaffold or coated onto thescaffold.

Additionally, other factors which influence the growth and/or activityof particular cells may also be used. For example, in the case ofchondrocytes, a factor such as a chondroitinase which stimulatescartilage production by chondrocytes can be added to the matrix in orderto maintain chondrocytes in a hypertrophic state as described in U.S.Patent Application No. 2002/0122790 incorporated herein by reference. Inanother aspect, the methods of the present invention include thepresence of polysulphated alginates or other polysulphatedpolysaccharides such as polysulphated cyclodextrin and/or polysulphatedinulin, or other components capable of stimulating production ofextracellular matrix of connective tissue cells as described inInternational Patent Publication No. WO 2005/054446 incorporated hereinby reference.

The cell or tissue to be repaired and/or regenerated may be contacted invivo or in vitro by any of the methods described herein. For example,the ECM compositions may be injected or implanted (e.g., via ECM tissue,a patch or coated device of the present invention) into the subject. Inanother aspect, the tissue or cells to be repaired and/or regeneratedmay be harvested from the subject and cultured in vitro and subsequentlyimplanted or administered to the subject using known surgicaltechniques.

As discussed above, the ECM compositions of the present invention may beprocessed in a variety of ways. Accordingly, in one embodiment, thepresent invention includes a tissue culture system. In various aspects,the culture system is composed of the extracellular matrix compositionsdescribed herein. The ECM compositions of the present invention may beincorporated into the tissue culture system in a variety of ways. Forexample, compositions may be incorporated as coatings, by impregnatingthree-dimensional scaffold materials as described herein, or asadditives to media for culturing cells. Accordingly, in one aspect, theculture system can include three-dimensional support materialsimpregnated with any of the extracellular matrix compositions describedherein, such as growth factors or embryonic proteins.

The extracellular matrix compositions described herein may serve as asupport or three-dimensional support for the growth of various celltypes. Any cell type capable of cell culture is contemplated. In oneaspect, the culture system can be used to support the growth of stemcells. In another aspect, the stem cells are embryonic stem cells,mesenchymal stem cells or neuronal stem cells.

The tissue culture system may be used for generating additional ECMcompositions, such as implantable tissue. Accordingly, culturing ofcells using the tissue culture system of the present invention may beperformed in vivo or in vitro. For example, the tissue culture system ofthe present invention may be used to generate ECM compositions forinjection or implantation into a subject. The ECM compositions generatedby the tissue culture system may be processed and used in any methoddescribed herein.

The ECM compositions of the present invention may be used as abiological vehicle for cell delivery. As described herein, the ECMcompositions may include both soluble and/or non-soluble fractions. Assuch, in another embodiment of the present invention, a biologicalvehicle for cell delivery or maintenance at a site of delivery includingthe extracellular matrix compositions of the present invention, isdescribed. The ECM compositions of the present invention, includingcells and three-dimensional tissue compositions, may be used to promoteand/or support growth of cells in vivo. The vehicle can be used in anyappropriate application, for example to support injections of cells,such as stem cells, into damaged heart muscle or for tendon and ligamentrepair as described above.

Appropriate cell compositions (e.g., isolated ECM cells of the presentinvention and/or additional biological agents) can be administeredbefore, after or during the ECM compositions are implanted oradministered. For example, the cells can be seeded into the site ofadministration, defect, and/or implantation before the culture system orbiological delivery vehicle is implanted into the subject.Alternatively, the appropriate cell compositions can be administeredafter (e.g., by injection into the site). The cells act therein toinduce tissue regeneration and/or cell repair. The cells can be seededby any means that allows administration of the cells to the defect site,for example, by injection. Injection of the cells can be by any meansthat maintains the viability of the cells, such as, by syringe orarthroscope.

Extracellular matrix compositions have been described for promotingangiogenesis in organs and tissues by administering such compositions topromote endothelialization and vascularization in the heart and relatedtissues. Accordingly, in yet another embodiment, the present inventionincludes a surface coating used in association with implantation of adevice in a subject including the extracellular matrix compositionsdescribed herein. The coating may be applied to any device used inimplantation or penetration of a subject, such as a pacemaker, a stent,a stent graft, a vascular prosthesis, a heart valve, a shunt, a drugdelivery port or a catheter. In certain aspects, the coating can be usedfor modifying wound healing, modifying inflammation, modifying a fibrouscapsule formation, modifying tissue ingrowth, or modifying cellingrowth. In another embodiment, the present invention includes a fortreatment of damaged tissue, such as heart, intestinal, infarcted orischemic tissue. Presented below are examples discussing generation ofextracellular matrix compositions contemplated for such applications.The preparation and use of extracellular matrix compositions grown undernormal oxygen conditions is described in U.S. Patent Application No.2004/0219134 incorporated herein by reference.

In another embodiment, the present invention includes a tissueregeneration patch including the extracellular matrix compositionsdescribed herein. In another embodiment the present invention includes amethod for improvement of a skin surface in a subject includingadministering to the subject at the site of a wrinkle, the extracellularmatrix compositions described herein. In yet another embodiment, thepresent invention includes a method for soft tissue repair oraugmentation in a subject including administering to the subject at thesite of a wrinkle, the extracellular matrix compositions describedherein. The preparation and use of extracellular matrix compositionscreated under normal oxygen culture conditions for the repair and/orregeneration of cells, improvement of skin surfaces, and soft tissuerepair are described in U.S. Pat. No. 5,830,708, U.S. Pat. No.6,284,284, U.S. Patent Application No. 2002/0019339 and U.S. PatentApplication No. 2002/0038152 incorporated herein by reference.

In another embodiment, the present invention includes a biologicalanti-adhesion agent including the extracellular matrix compositionsdescribed herein. The agent can be used in such applications asanti-adhesion patches used after the creation of intestinal or bloodvessel anastomises.

The compositions or active components used herein, will generally beused in an amount effective to treat or prevent the particular diseasebeing treated. The compositions may be administered therapeutically toachieve therapeutic benefit or prophylactically to achieve prophylacticbenefit. By therapeutic benefit is meant eradication or amelioration ofthe underlying condition or disorder being treated. Therapeutic benefitalso includes halting or slowing the progression of the disease,regardless of whether improvement is realized.

The amount of the composition administered will depend upon a variety offactors, including, for example, the type of composition, the particularindication being treated, the mode of administration, whether thedesired benefit is prophylactic or therapeutic, the severity of theindication being treated and the age and weight of the patient, andeffectiveness of the dosage form. Determination of an effective dosageis well within the capabilities of those skilled in the art.

Initial dosages may be estimated initially from in vitro assays. Initialdosages can also be estimated from in vivo data, such as animal models.Animals models useful for testing the efficacy of compositions forenhancing hair growth include, among others, rodents, primates, andother mammals. The skilled artisans can determine dosages suitable forhuman administration by extrapolation from the in vitro and animal data.

Dosage amounts will depend upon, among other factors, the activity ofthe conditioned media, the mode of administration, the condition beingtreated, and various factors discussed above. Dosage amount and intervalmay be adjusted individually to provide levels sufficient to themaintain the therapeutic or prophylactic effect.

In one embodiment, the methods of the invention include administrationof the ECM in combination with an anti-inflammatory agent,antihistamines, chemotherapeutic agent, immunomodulator, therapeuticantibody or a protein kinase inhibitor, to a tissue, cell or subject inneed of such treatment. While not wanting to be limiting,chemotherapeutic agents include antimetabolites, such as methotrexate,DNA cross-linking agents, such as cisplatin/carboplatin; alkylatingagents, such as canbusil; topoisomerase I inhibitors such asdactinomicin; microtubule inhibitors such as taxol (paclitaxol), and thelike. Other chemotherapeutic agents include, for example, a vincaalkaloid, mitomycin-type antibiotic, bleomycin-type antibiotic,antifolate, colchicine, demecoline, etoposide, taxane, anthracyclineantibiotic, doxorubicin, daunorubicin, carminomycin, epirubicin,idarubicin, mithoxanthrone, 4-dimethoxy-daunomycin,11-deoxydaunorubicin, 13-deoxydaunorubicin, adriamycin-14-benzoate,adriamycin-14-octanoate, adriamycin-14-naphthaleneacetate, amsacrine,carmustine, cyclophosphamide, cytarabine, etoposide, lovastatin,melphalan, topetecan, oxalaplatin, chlorambucil, methtrexate, lomustine,thioguanine, asparaginase, vinblastine, vindesine, tamoxifen, ormechlorethamine While not wanting to be limiting, therapeutic antibodiesinclude antibodies directed against the HER2 protein, such astrastuzumab; antibodies directed against growth factors or growth factorreceptors, such as bevacizumab, which targets vascular endothelialgrowth factor, and OSI-774, which targets epidermal growth factor;antibodies targeting integrin receptors, such as Vitaxin (also known asMEDI-522), and the like. Classes of anticancer agents suitable for usein compositions and methods of the present invention include, but arenot limited to: 1) alkaloids, including, microtubule inhibitors (e.g.,Vincristine, Vinblastine, and Vindesine, etc.), microtubule stabilizers(e.g., Paclitaxel [Taxol], and Docetaxel, Taxotere, etc.), and chromatinfunction inhibitors, including, topoisomerase inhibitors, such as,epipodophyllotoxins (e.g., Etoposide [VP-16], and Teniposide [VM-26],etc.), and agents that target topoisomerase I (e.g., Camptothecin andIsirinotecan [CPT-11], etc.); 2) covalent DNA-binding agents [alkylatingagents], including, nitrogen mustards (e.g., Mechlorethamine,Chlorambucil, Cyclophosphamide, Ifosphamide, and Busulfan [Myleran],etc.), nitrosoureas (e.g., Carmustine, Lomustine, and Semustine, etc.),and other alkylating agents (e.g., Dacarbazine, Hydroxymethylmelamine,Thiotepa, and Mitocycin, etc.); 3) noncovalent DNA-binding agents[antitumor antibiotics], including, nucleic acid inhibitors (e.g.,Dactinomycin [Actinomycin D], etc.), anthracyclines (e.g., Daunorubicin[Daunomycin, and Cerubidine], Doxorubicin [Adriamycin], and Idarubicin[Idamycin], etc.), anthracenediones (e.g., anthracycline analogues, suchas, [Mitoxantrone], etc.), bleomycins (Blenoxane), etc., and plicamycin(Mithramycin), etc.; 4) antimetabolites, including, antifolates (e.g.,Methotrexate, Folex, and Mexate, etc.), purine antimetabolites (e.g.,6-Mercaptopurine [6-MP, Purinethol], 6-Thioguanine [6-TG], Azathioprine,Acyclovir, Ganciclovir, Chlorodeoxyadenosine, 2-Chlorodeoxyadenosine[CdA], and 2′-Deoxycoformycin [Pentostatin], etc.), pyrimidineantagonists (e.g., fluoropyrimidines [e.g., 5-fluorouracil (Adrucil),5-fluorodeoxyuridine (FdUrd) (Floxuridine)] etc.), and cytosinearabinosides (e.g., Cytosar [ara-C] and Fludarabine, etc.); 5) enzymes,including, L-asparaginase; 6) hormones, including, glucocorticoids, suchas, antiestrogens (e.g., Tamoxifen, etc.), nonsteroidal antiandrogens(e.g., Flutamide, etc.), and aromatase inhibitors (e.g., anastrozole[Arimidex], etc.); 7) platinum compounds (e.g., Cisplatin andCarboplatin, etc.); 8) monoclonal antibodies conjugated with anticancerdrugs, toxins, and/or radionuclides, etc.; 9) biological responsemodifiers (e.g., interferons [e.g., IFN-.alpha., etc.] and interleukins[e.g., IL-2, etc.], etc.); 10) adoptive immunotherapy; 11) hematopoieticgrowth factors; 12) agents that induce tumor cell differentiation (e.g.,all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14)antisense therapy techniques; 15) tumor vaccines; 16) therapies directedagainst tumor metastases (e.g., Batimistat, etc.); and 17) inhibitors ofangiogenesis.

The pharmaceutical composition and method of the present invention mayfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of the above mentionedpathological conditions. Examples of other therapeutic agents includethe following: cyclosporins (e.g., cyclosporin A), CTLA4-Ig, antibodiessuch as ICAM-3, anti-IL-2 receptor (Anti-Tac), anti-CD45RB, anti-CD2,anti-CD3 (OKT-3), anti-CD4, anti-CD80, anti-CD86, agents blocking theinteraction between CD40 and gp39, such as antibodies specific for CD40and/or gp39 (i.e., CD154), fusion proteins constructed from CD40 andgp39 (CD40Ig and CD8 gp39), inhibitors, such as nuclear translocationinhibitors, of NF-kappa B function, such as deoxyspergualin (DSG),cholesterol biosynthesis inhibitors such as HMG CoA reductase inhibitors(lovastatin and simvastatin), non-steroidal antiinflammatory drugs(NSAIDs) such as ibuprofen and cyclooxygenase inhibitors such asrofecoxib, steroids such as prednisone or dexamethasone, gold compounds,antiproliferative agents such as methotrexate, FK506 (tacrolimus,Prograf), mycophenolate mofetil, cytotoxic drugs such as azathioprineand cyclophosphamide, TNF-a inhibitors such as tenidap, anti-TNFantibodies or soluble TNF receptor, and rapamycin (sirolimus orRapamune) or derivatives thereof.

Other agents that may be administered in combination with inventioncompositions and methods including protein therapeutic agents such ascytokines, immunomodulatory agents and antibodies. As used herein theterm “cytokine” encompasses chemokines, interleukins, lymphokines,monokines, colony stimulating factors, and receptor associated proteins,and functional fragments thereof. As used herein, the term “functionalfragment” refers to a polypeptide or peptide which possesses biologicalfunction or activity that is identified through a defined functionalassay.

The cytokines include endothelial monocyte activating polypeptide II(EMAP-II), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF),macrophage-CSF (M-CSF), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, andIL-13, interferons, and the like and which is associated with aparticular biologic, morphologic, or phenotypic alteration in a cell orcell mechanism.

When other therapeutic agents are employed in combination with thecompositions or methods of the present invention they may be used forexample in amounts as noted in the Physician Desk Reference (PDR) or asotherwise determined by one having ordinary skill in the art.

Presented below are examples discussing generation of extracellularmatrix compositions contemplated for the discussed applications. Thefollowing examples are provided to further illustrate the embodiments ofthe present invention, but are not intended to limit the scope of theinvention. While they are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart may alternatively be used.

EXAMPLE 1 Differential Gene Expression in ECM Compositions Grown UnderHypoxic Conditions

Primary human neonatal foreskin fibroblasts were cultured as standardmonolayers in tissue culture flasks and compared to three-dimensionalfibroblast cultures, within a naturally deposited, fetal-like ECM. Thecultures were grown as disclosed herein. To assess differentialexpression of genes, samples of total RNA were completed using AgilentWhole Human Genome Oligo Microarrays® for global gene expression(including less than 40,000 genes) following the manufacturer'sprotocol.

Upon comparison, fibroblasts were found to regulate collagen and ECMgene expression in three-dimensional cultures within a hypoxic culturednaturally secreted ECM. Upregulation and downregulation of expression ofvarious collagen and ECM genes are evident in Table 3.

TABLE 3 Differential Collagen and ECM Expression in HypoxicThree-Dimensional Fibroblast Cultures GENE FOLD INCREASE FOLD DECREASECOL4A1 17.2 COL20A1 6.88 COL19A1 5.22 COL9A1 4.81 COL10A1 4.45 COL6A33.48 COL9A2 2.48 COL14A1 2 SPARC 2.74 COL1A2 3.45 COL13A1 4 COL18A1 4.76COL1A2 7.14

Upon comparison, fibroblasts were found to regulate gene expression ofWnt pathway genes in three-dimensional cultures within a hypoxiccultured naturally secreted ECM. Upregulation and downregulation ofexpression of various Wnt pathway genes are evident in Table 4.

TABLE 4 Differential Wnt Expression in Hypoxic Three-dimensionalFibroblast Cultures GENE FOLD INCREASE FOLD DECREASE WNT 4 5.94 WNT 7a5.43 WNT 7b 4.05 WNT 2b 3.95 WNT 10a 3.86 WNT 8b 3.48 WNT 6 3.36 WNT 3a3.19 WNT 9b 3.06 WNT 9a 3.02 WNT 11 2.89 WNT 5a 8.33 WNT 2 7.14 WNT 5b5.26 LRP6 3.43 LRP3 2.27 LRP11 10 LRP12 7.69 DKK1 50 DKK3 5.88 FSZD54.48 FRZ9 3.85 FRZB 3.36 FRZD1 2.94 SFRP2 2.95 FRZD1 2.92 FRZD3 2.84AXIN2 4.4 KREMEN2 4.24 KREMEN1 3.45 b-CATENIN 4.76 GSK3b 11.1 GSK3a 6.67bFGF 50

Upon comparison, fibroblasts were found to regulate gene expression ofbone morphogenetic protein (BMP) pathway genes in three-dimensionalcultures within a hypoxic cultured naturally secreted ECM. Upregulationand downregulation of expression of various BMP pathway genes areevident in Table 5.

TABLE 5 Differential BMP Expression in Hypoxic Three-DimensionalFibroblast Cultures GENE FOLD INCREASE FOLD DECREASE BMP7/OP1 4.88 BMP24.19 BMP5 3.49 BMP3 3.44 BMPrecIb 3.37 BMP8b 3.36 BMP 8a 3.15 BMP 102.86 BMP1 2.12 BMPrecIa 2.5 Osteocalcin 2.5 Osteopontin 6.25 BMPrecII6.25

Upon comparison, fibroblasts were found to regulate expression ofadditional genes in three-dimensional cultures within a the hypoxiccultured naturally secreted ECM. Upregulation and downregulation ofexpression of additional genes are evident in Table 6.

TABLE 6 Additional Gene Expression Changes Resulting from Low OxygenCulture of Fibroblast ECM In Vivo. GENES FOLD INCREASE FOLD DECREASECollagens COL5A1 6.21 COL9A2 3.96 COL6A2 3.78 COL6A2 3.21 COL11A1 3.07COL8A1 2.78 COL4A5 2.45 COL7A1 2.45 COL18A1 2.41 COL12A1 2.04 COL1A2 0.5COL14A1 0.45 COL4A1 0.45 COL5A2 0.23 COL6A1 0.16 MatrixMetalloproteinases (MMPs) MMP23B 2.75 MMP27 0.24 MMP28 0.17 MMP10 0.16MMP1 0.16 MMP7 0.1 MMP14 0.08 MMP3 0.06 MMP12 0.05 Other ECM HAPLN3 8.11ACAN L12234 6.48 AGC1 3.32 LAMA3 2.92 LAMA1 2.14 LAMA5 2.14

EXAMPLE 2 Production of Hypoxic ECM Using Primary Human NeonatalForeskin Fibroblasts

Two examples are provided for hypoxic culture of ECM using primary humanneonatal foreskin fibroblasts.

Primary human neonatal foreskin fibroblasts were expanded in tissueculture flasks in the presence of 10% fetal bovine serum, 90% HighGlucose DMEM with 2 mM L-glutamine (10% FBS/DMEM). Cells weresubcultured using 0.05% trypsin/EDTA solution until the 3^(rd) passageat which time they were seeded to either Cytodex-1 dextran beads at 0.04mgs dry beads/ml of medium (5e⁶ cells/10 mgs beads in a 125 ml spinnerflask filled with 100-120 mls), or to nylon mesh (25e⁶ cells/6×100 cm²nylon). All cultures were kept in normal atmosphere and 5% CO₂ forexpansion and seeding, at which point low oxygen cultures were split toan airtight chamber which was flooded with 95% nitrogen/5% CO₂ so that ahypoxic environment could be created within the culture medium. Thissystem is maintains about 1-5% oxygen within the culture vessel. Cellswere mixed well into the minimum volume needed to cover nylon or beadsfor seeding, and were subsequently mixed once after 30 minutes, thenallowed to sit overnight in a humidified 37° C. incubator. Cultures werefed 10% FBS/DMEM for 2-4 weeks with a 50-70% media exchange, every 2-3days while cells proliferated and then began depositing ECM. Cultureswere fed for another 4-6 weeks using 10% bovine calf serum with ironsupplement, and 20 ug/ml ascorbic acid in place of FBS. Spinner flaskswere mixed at 15-25 rpm initially and for about 2-4 weeks, at which timethey were increased to 45 rpm and maintained at this rate thereafter.Bead cultures formed large amorphous structures containing ECM of asmuch as 0.5 to 1.0 cm in width and diameter after 4 weeks, and thesecultures were therefore hypoxic due to gas diffusion and high metabolicrequirements.

In an additional example, primary human neonatal foreskin fibroblastswere expanded in monolayer flasks, and then cultured on nylon meshscaffolds to support development of an extracellular matrix (ECM) invitro. Fibroblasts were expanded in DMEM with high glucose, 2 mML-glutamine, and 10% (v/v) fetal bovine serum. Cultures were alsosupplemented with 20 μg/ml ascorbic acid. After 3 weeks in ambientoxygen (approximately 16%-20% oxygen) duplicate ECM-containing cultureswere switched to hypoxic culture conditions (1%-5% oxygen) in a sealedchamber flushed extensively with 95% nitrogen/5% carbon dioxide(Cat.#MC-101, Billups-Rothenberg, Inc., Del Mar, Calif.). To ensuredepletion of atmospheric oxygen from the culture medium, 2-3 hours laterthe atmosphere was replaced to ensure that the medium containedapproximately 1-3% oxygen. Both sets of ECM-containing cultures weregrown with twice weekly feedings for another 4 weeks, and then cultureswere prepared for RNA isolation. Total cellular RNA was isolated using acommercially available kit according to he manufacturers instructions(Cat.#Z3100, Promega, Inc.). Purified RNA samples were stored at −80°C., prior to processing for microarray analysis of gene expression usingAgilent Whole Human Genome Oligo Microarrays®.

In analyzing the results, there were approximately 5,500 differentiallyexpressed transcripts detected from probes prepared from ambient oxygenin comparison to probes from low oxygen cultures, using Agilent WholeHuman Genome Oligo Microarrays®. Of these, about half (2,500) weregreater than 2.0 fold increased by low oxygen, and about half (2,500)were decreased greater than 2.0 fold in low oxygen. This indicates thatlow oxygen led to significant changes in gene expression in vitro. Ofparticular interest, transcripts for ECM proteins, particularly a numberof collagen genes were up-regulated, while a number of genes formatrix-degrading enzymes were down-regulated.

EXAMPLE 3 Tissue-Engineered Human Embryonic Extracellular Matrix forTherapeutic Applications

The embryonic extracellular matrix (ECM) creates an environmentconducive to rapid cell proliferation and healing without the formationof scars or adhesions. It was hypothesized that the growth of humanneonatal fibroblasts in 3 dimensions under conditions that simulate theearly embryonic environment prior to angiogenesis (hypoxia and reducedgravitational forces) would generate an ECM with fetal properties. Genechip array analysis showed the differential expression of over 5000genes under the hypoxic versus traditional tissue culture conditions.The ECM produced was similar to fetal mesenchymal tissue in that it isrelatively rich in collagens type III, IV, and V, and glycoproteins suchas fibronectin, SPARC, thrombospondin, and hyaluronic acid. Since theECM also plays an important regulatory role in binding and presentinggrowth factors in putative niches which support regenerative stem cellpopulations with key growth factors, we evaluated the effects of hypoxiaon growth factor expression during the development of the fetal-like ECMin culture. Hypoxia can also enhance expression of factors whichregulate wound healing and organogenesis, such as VEGF, FGF-7, andTGF-β, as well as multiple Wnt's including Wnt's 2b,4,7a,10a, and 11.The embryonic human ECM also stimulated an increase of metabolicactivity in human fibroblasts in vitro, as measured by increasedenzymatic activity using the MTT assay. Additionally, we detected anincrease in cell number in response to human ECM. This human ECM can beused as a biological surface coating, and tissue filler treatment invarious therapeutic applications where new tissue growth and healingwithout scarring or adhesions.

EXAMPLE 4 Production of Naturally-Soluble WNT Activity for RegenerativeMedicine Applications

Stem or progenitor cells that can regenerate adult tissues, such as skinor blood, recapitulate embryonic development to some extent toaccomplish this regeneration. A growing number of studies have shownthat key regulators of stem cell pluripotency and lineage-specificdifferentiation active during embryogenesis are re-expressed in theadult under certain circumstances. The Wnt family of secretedmorphogenetic growth and development factors is among the growth factorswhich can potentially provide valuable research tools and eventuallytherapeutic treatments in the clinic. However, Wnt's have provenrefractory to standard recombinant expression and purificationtechniques to date on a commercial scale, and there are no reports oflarge-scale Wnt protein production to enable clinical development ofWnt-based products. Techniques have been developed for growingfetal-like ECM in culture using neonatal human dermal fibroblasts onvarious scaffolds in culture to generate three-dimensionaltissue-equivalents. In this process, it was discovered that thesecultures can provide a commercial-scale source of bioactive Wnt'scontained in the serum-free conditioned medium used for ECM production.Here we present data on this Wnt product candidate.

Gene expression analysis of the cells demonstrated that at least 3 WNTgenes were expressed (Wnt 5a, Wnt 7a, and Wnt 11), and a small number ofgenes related to Wnt signaling were expressed as well; however, theirfunction is not completely understood. The gene expression data wasextended to an in vitro bioassay for Wnt-signaling (nucleartranslocation of β-catenin in primary human epidermal keratinocytes) andWnt activity on blood stem cells was evaluated. Both assays demonstratedactivity consistent with canonical Wnt activity. Furthermore,conditioned media from these cultures showed Wnt activity when injectedinto the skin of mice, inducing hair follicle stem cells to enteranagen, thus causing hair growth. This indicates that the stabilized Wntactivity within the defined and serum-free condition medium did notrequire purification. This product can be used for hair follicleregeneration and as a valuable research tool for the culture of varioushuman stem cells.

EXAMPLE 5 Hypoxic Fibroblasts Demonstrate Unique ECM Production andGrowth Factor Expression

Human neonatal dermal fibroblasts produce an ECM when cultured in vitro,which closely mimics the dermis and which can replace the damaged dermisin regenerative medicine applications such as wound healing. Since theprocess of wound healing also recapitulates embryonic development, bysimulating the embryonic environment we hypothesize that the ECMproduced will provide an enhanced ECM for tissue regenerationapplications. Therefore, human neonatal fibroblast-derived ECM weregrown under hypoxic conditions in culture, to simulate the hypoxia whichexists in the early embryo prior to angiogenesis. The goal was togenerate an ECM with fetal properties using hypoxic conditions duringtissue development in culture.

The ECM produced in these hypoxic cultures was similar to fetalmesenchymal tissue in that it is relatively rich in collagens type IIIand V, and glycoproteins such as fibronectin, SPARC, thrombospondin, andhyaluronic acid. Since the ECM also plays an important regulatory rolein binding and presenting growth factors in putative niches whichsupport regenerative stem cell populations with key growth factors, weevaluated the effects of hypoxia on growth factor expression during thedevelopment of the fetal-like ECM in culture. It was shown that hypoxiacan also enhance expression of factors which regulate wound healing andorganogenesis, such as VEGF, FGF-7, and TGF-β.

The human ECM also stimulated an increase of metabolic activity in humanfibroblasts in vitro, as measured by increased enzymatic activity usingthe MTT assay. Additionally, an increase in cell number in response tohuman ECM was detected. These results support the use of this human ECMas a coating/scaffold in embryonic cell cultures and as a biologicalsurface coating/filler in various therapeutic applications or medicaldevices.

EXAMPLE 6 Extracellular Matrix Compositions in Melanoma, Glioma, andBreast Cancer

Tumor growth of B16 (melanoma cell line), C6 (glioma cell line), and MDA435 (breast cancer cell line) cells, in the presence or absence of ECM,and with or without cisplatin, were examined in a chorioallantoicmembrane of the fertilized chicken egg assay (CAM). Briefly, eggs werecandled for viability. The air sack was displaced creating a blisterbetween the shell and chorioallantoic membranes at the equator of theegg. A window was cut through the shell to gain access to the CAM. Cellswere added to the CAM as follows: B16 cells, 5 million per egg; C6cells, 5 million per egg; and MD435 cells, 5 million per egg. In theeggs treated with ECM, the ECM (˜100 μg/treatment) and cells were addedto the eggs at the same time. In the eggs treated with cisplatin, thecisplatin (250 μL of 1 mg/mL cisplatin solution) was added topically thenext day. The eggs were incubated for 10 days at which time the tumorswere removed and weighed.

Tumor mass was significantly reduced by the presence of ECM. FIGS. 1 and2 show that the weight of tumors produced by B16 cells was reduced inthe presence of ECM versus the absence of ECM. The weight of tumors wasalso reduced whether tumor cells were mixed with ECM or grown on ECM(FIG. 3). FIGS. 4 and 5 show that the weight of tumors produced by C6cells was reduced in the presence of ECM versus no treatment. Moreover,tumor weight was further reduced in the presence of ECM with cisplatin.FIG. 7 shows that the weight of tumors produced by MDA 435 breast cancercells was reduced in the presence of ECM versus no treatment and wasfurther reduced by ECM with cisplatin.

In another CAM assay, B16 cells were injected into eggs (2-3millioncells/egg) with a 100 μL dose of BioNeusis (10 mg/mL). BioNeusis is themedium conditioned by the fibroblasts in the bioreactor used to generateECM and is very similar to the ECM only in liquid form. FIG. 16 showsthe tumor growth in the CAM assay was reduced in the presence ofBioNeusis as compared to no treatment. In a further study, B16 cellswere grown in the presence of increasing concentrations of liquid ECM,with growth reduced in all concentrations of ECM (FIG. 17). In anotherstudy, B16 cells were grown in liquid ECM of various molecular weightfractions as compared to B16 cells grown in unfractionated liquid ECM inthe CAM assay. All molecular weight fractions except the >100,000 daltonfraction showed an increase in the percent reduction of tumor mass ascompared to unfractionated ECM (FIG. 18).

Tumor growth of B16 cells, C6 cells, or MDA435 cells in the presence orabsence of ECM, and with or without cisplatin, were examined in nudemice. Cells, ECM and Cisplatin were injected simultaneouslysubcutaneously in the lower haunch of a nude mouse. Tumor growth wasfollowed over time using photography, measurements of length and width,then finally by weight. FIGS. 8 and 9 show the growth of tumors from C6cells in nude mice in the presence (FIG. 9) and absence (FIG. 8) of ECMon day 14 post-injection. FIGS. 10 and 11 show the growth of tumors fromC6 cells in nude mice in the presence of ECM with cisplatin (FIG. 10)and cisplatin without ECM (FIG. 11) of ECM on day 14 post-injection.Cells grown with ECM showed reduced or no tumor growth, in comparison tothe control (no treatment) which did form palpable tumors. FIG. 12 showsa plot of the tumor growth over 18 days in which mice treated with ECMshowed reduced tumor growth, as compared to the no treatment control andmice treated with cisplatin, with or without ECM showed no growth. In ananalogous study, MDA435 cells grown with ECM (FIG. 13B), with ECM pluscisplatin (FIG. 13C), or with cisplatin alone (FIG. 13D) showed reducedor no tumor growth, in comparison to the no treatment control (FIG.13A), which did form palpable tumors. FIG. 14 shows a plot of the tumorgrowth over 25 days in which mice treated with ECM showed reduced tumorgrowth, as compared to the no treatment control. Mice treated withcisplatin, with or without ECM showed a reduction in tumor mass to zero.FIG. 15 shows the results of B16 cells grown in nude mice with ECM(right side) or without (left side) ECM, in which tumor growth wasinhibited in the presence of ECM.

EXAMPLE 7 Inhibition of Metastasis of Adenocarcinoma Using ExtracellularMatrix Compositions

Metastasis of primary tumors of HCT cells (human colon adenocarcinomacell line), in the presence and absence of ECM (both insoluble (iECM)and soluble (sECM) fractions), were examined in a chorioallantoicmembrane of the fertilized chicken egg assay (CAM). Briefly, eggs wereprocessed as previously described. Tumors were formed by subsequentexposure to HCT cells and incubation. Samples were stained withfluorescently labeled anti-CD44+ antibodies (green) and examined byfluorescence microscopy. In the control sample (no ECM treatment), highnumbers of cancer cells were observed migrating out of the metastasizingprimary tumor mass and entering circulation. In samples treated witheither iECM or sECM, metastasis was greatly inhibited as few or nocancer cells were observed migrating out of primary tumor masses.

Although the invention has been described with reference to the aboveexamples and Attachment 1, the entire contents of which are incorporatedherein by reference, it will be understood that modifications andvariations are encompassed within the spirit and scope of the invention.Accordingly, the invention is limited only by the following claims.

1-11. (canceled)
 12. A method of treating cancer in a subject comprisingadministering an ECM to the subject wherein the ECM is produced by themethod comprising culturing fibroblast cells on microbeads under hypoxicconditions of 1-5% oxygen to produce a hypoxic ECM composition, therebythreating the cancer.
 13. The method of claim 12, wherein the cancer isselected from the group consisting of melanoma, glioma, andadenocarcinoma, bladder cancer, bone cancer, cancer of the bone marrow,brain cancer, spinal cancer, breast cancer, cervical cancer, gallbladder cancer, cancer of the ganglia, cancer of the gastrointestinaltract, stomach cancer, colon cancer, cancer of the heart, kidney cancer,liver cancer, lung cancer, muscle cancer, ovarian cancer, pancreaticcancer, parathyroid cancer, cancer of the penis, prostate cancer, cancerof the salivary glands, skin cancer, cancer of the spleen, cancer of thetestis, thymus cancer, thyroid cancer, or uterine cancer.
 14. The methodof claim 12, wherein, wherein the hypoxic ECM composition has a solublefraction.
 15. The method of claim 12, wherein the hypoxic ECMcomposition has a non-soluble fraction.
 16. The method of claim 12,wherein the hypoxic ECM composition is a combination of a soluble andnon-soluble fraction.
 17. The method of claim 14, wherein the solublefraction comprises a molecular weight fraction comprising moleculeshaving a molecular weight less than 30,000 Daltons.
 18. The method ofclaim 17, wherein the molecules have a molecular weight less than 5,000Daltons.
 19. The method of claim 12, further comprising administratingto the subject an anti-inflammatory agent, antihistamines,chemotherapeutic agent, immunomodulator, therapeutic antibody or aprotein kinase inhibitor.
 20. The method of claim 19, wherein thechemotherapeutic agent is an alkaloid; covalent DNA-binding agent;noncovalent DNA-binding agent; antimetabolite; enzyme; hormone; platinumcompound; monoclonal antibody conjugated with anticancer a drug, toxin,and/or radionuclide; biological response modifier; hematopoietic growthfactor; inhibitor of angiogenesis; DNA cross-linking agent;topoisomerase I inhibitor; or microtubule inhibitor.
 21. The method ofclaim 19, wherein the chemotherapeutic agent is selected from the groupconsisting of 2-Chlorodeoxyadenosine, 2′-Deoxycoformycin,2′-Deoxycoformycin, 5-fluorouracil, 5-fluorodeoxyuridine,4-dimethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin,adriamycin-14-benzoate, adriamycin-14-octanoate,adriamycin-14-naphthaleneacetate, 6-Mercaptopurine, 6-Thioguanine,acyclovir, amsacrine, anastrozole, asparaginase, azathioprine, busulfan,camptothecin, canbusil, carboplatin, carminomycin, carmustine,chlorambucil, chlorodeoxyadenosine, cisplatin, cyclophosphamide,cytarabine, dactinomycin, dacarbazine, daunorubicin, doxorubicin,docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, flutamide,hydroxymethylmelamine, idarubicin, ifosphamide, isirinotecan, lomustine,lovastatin, mechlorethamine, melphalan, methotrexate, mithoxanthrone,mitocycin, oxalaplatin, semustine, thiotepa, tamoxifen, taxol, taxotere;teniposide, thioguanine, topetecan, vincristine, vinblastine, andvindesine.
 22. The method of claim 12, wherein the subject had aprevious surgical resection.
 23. The method of claim 12, wherein thehypoxic ECM further comprises a chemotherapeutic agent and/or animmunomodulatory agent.
 24. The method of claim 23, wherein thechemotherapeutic agent is selected from the group consisting of2-Chlorodeoxyadenosine, 2′-Deoxycoformycin, 2′-Deoxycoformycin,5-fluorouracil, 5-fluorodeoxyuridine, 4-dimethoxy-daunomycin,11-deoxydaunorubicin, 13-deoxydaunorubicin, adriamycin-14-benzoate,adriamycin-14-octanoate, adriamycin-14-naphthaleneacetate,6-Mercaptopurine, 6-Thioguanine, azathioprine, acyclovir, amsacrine,anastrozole, asparaginase, busulfan, camptothecin, canbusil,carboplatin, carminomycin, carmustine, chlorambucil,chlorodeoxyadenosine, cisplatin, cyclophosphamide, cytarabine,dactinomycin, dacarbazine, daunorubicin, doxorubicin, docetaxel,doxorubicin, epirubicin, etoposide, fludarabine, flutamide,hydroxymethylmelamine, idarubicin, ifosphamide, isirinotecan, lomustine,lovastatin, mechlorethamine, melphalan, methotrexate, mithoxanthrone,mitocycin, oxalaplatin, semustine, thiotepa, tamoxifen, taxol, taxotere;teniposide, thioguanine, topetecan, vincristine, vinblastine, andvindesine.
 25. The method of claim 12, wherein the fibroblast cells arehuman.
 26. The method of claim 12, wherein the fibroblast cells areadult, fetal, neonatal or embryonic fibroblast cells.